Serving in the military leads to mental diseases, such as post-traumatic stress disorder (PTSD), for a percentage of soldiers globally. The number of veterans with PTSD is increasing and, although medication and psychological treatments are offered, treatment results could be improved. Historically, different forms of nature-based therapy have been used for this target group. However, in spite of anecdotally good results, studies measuring the effect of this form of therapy are still lacking. The aim of this study is to explore how veterans with PTSD manage their everyday lives during and after a ten-week nature-based intervention in a therapy garden. Methods: Eight veterans participated in qualitative interviews, which were conducted during a one-year period and were analyzed using interpretative phenomenological analysis (IPA). Results: Five themes emerged from the IPA analysis: Bodily symptoms; relationships; building new identities; the future; and lessons learned. All the participating veterans gained a greater insight into and mastering of their condition, achieved better control of their lives, and developed tools to handle life situations more appropriately and to build a new identity. This improved their ability to participate in social activities and employment. Conclusion: The results should be considered in the future treatment of veterans with PTSD. Historically, nature has been used as part of a healing process for soldiers who have been mentally wounded in World War l. The first systematic use of nature in the shape of horticultural activities was developed during World War I ,2 for soDiagnostic and Statistical Manual of Mental Disorders (DSM-V)  It has been the most stressful thing in my whole life. I just hope that I will be treated as I deserve.”The medical examinations did not lead to a physical explanation for their symptoms. Some began to consider whether there was a connection between their physical problems and their mental state and contacted the military health service. Others said that they continued to search for a physical diagnosis. A few described it as a struggle with large personal costs; it was no longer a question of getting a diagnosis, but of being respected as a person.“My dreams… I have positive dreams now. I still have some of those dreams that are hard to handle, but I am more distanced from them. Like standing outside and looking down on it. It does not affect me in the same way. More relaxed, you can say.”During the last five weeks of the intervention, all the veterans described changes in their bodily symptoms. These changes occurred at a different pace and did not always lead to a complete absence of symptoms. However, the veterans talked about how even a small change led to the realisation of how their symptoms had influenced their daily lives. In the third interview (toward the end of the intervention), one of the veterans mentioned that he had been able to sleep through the night for the first time in 18 years. Others talked about better quality of sleep, or about managing to let go of the anxiety connected with falling asleep and the nightmares the sleep might bring:“All that yoga and stuff, I do it because I am looking forward to lying down and listening  … I love to be in that trance-like state…I’m almost counting down.”The veterans said that the feelings of turmoil and of being in a state of constantly heightened alertness was reduced during the nature-based intervention. Most of the veterans experienced a peaceful feeling in their bodies during the nature-based activities. One said that it was as if breathing had become easier and this had led to reduced tension in his muscles. Another mentioned that this relaxed condition was a big motivator for doing some of the activities that he liked less:“I feel like the body is relaxing more. …normally I feel like a radar for people’s anger, even though it doesn’t have anything to do with me. If I go down the street and see someone who is angry, my system is up high [..]. Now I can tell myself it has nothing to do with me.”In the second interview , all the veterans stressed that the breathing techniques had become a useful tool for coping with stressful situations, such as sitting close to other people on a bus or going shopping in a crowded supermarket. They also told that they used to be very sensitive to other people’s emotions, but that this had changed during the intervention. One of the veterans described this change in the third interview:This change in behavioural patterns made it possible for the veterans to participate in social activities.“Your comrades are the ones you would die for…and the ones who help you handle your problems.”Two types of relationships were described as being affected by the veterans’ condition: Relationships with soldier comrades, and relationships with family and friends. The veterans described in detail the close relationships they had with comrades that had developed during service. They had felt a responsibility toward each other in their everyday lives, which had given them a sense of safety in spite of living with the constant threat of attack. The veterans described this fellowship with other soldiers in the first interview:“I feel guilty about being alive, and that I did not bring my comrade back home with me, even though I promised his mum I would.”When a comrade became wounded or died, the veterans experienced grief and guilt because they were alive. One of the veterans expressed it as follows:This veteran said that he had taken on a personal responsibility and that he blamed himself for his friend’s death. The fact that he had promised his friend’s mother that he would bring his friend home made it even worse. Two of the participants were sent home for treatment before they had finished their posting and recounted how they felt ashamed about leaving their comrades behind.“That guy, is he really sick?”Being on sick leave stimulated thoughts about what others might think of them, and made them feel that they were seen as cheats who had invented the symptoms themselves. Some felt it as if other people were talking badly about them behind their backs:“I haven’t got the room inside me to be there for her every day… I can’t… for me feelings are just not like before.”In the first interview, most of the veterans described a feeling of loneliness after coming home, and some felt an urge to isolate themselves from their relatives and friends. They said that during service, they considered their comrades to be replacements for their families. When they first returned home, their families could not replace this relationship. The veterans described their relationships with their families as difficult and often contradictory; on the one hand, their families were the most important thing in the world to them, while on the other hand, they could not handle being together with them. All the veterans thought that they had changed and were different from how they had been before service, and they found it difficult to meet their families’ needs for affection. One veteran, when talking about his girlfriend, who used to call him or send him messages every day, said the following:“I look at her…her hands, if she is carrying something, keys or… or if something looks different. If her hand is closed, I have to follow her movements until I have seen what she has got there.”In the first interview, the veterans also emphasised how they had changed after they had served; they were now in a constant state of alertness and unable to refrain from observing people’s behaviour when in a crowd. They said that they always felt the need to check for snipers and hide under tables when hearing the sound of airplanes. This behaviour frightened other people and was seen as being extreme. They felt that it had a significant effect on their ability to take part in social arrangements and it also led to unintended suspicion from their families. One veteran related the following episode about his wife coming home from work:“It has been terrible. Especially for my son. He has his needs… [interviewer: And you can`t give this to him?] “No, I can’t take it [touching and skin-contact].”All the veterans described how both physical contact and being able to sense affection were hard after coming home. One of them was unable to pick up his little son:“It’s hard for me to express how I’m feeling and maybe also hard for them to know what to say to me [...] Not even my family knows much about how I’m doing. It stays inside me, and it’s very very hard to open up and talk about how I’m feeling and thinking.”They also said that they experienced difficulties sharing feelings and talking about how they felt, which made their emotional communication with their relatives difficult:“If I had started in another group, where people had PTSD but for civil reasons, I don’t think it would have been the same… we have this mutual understanding [...] There are things we don’t need to say… we know what it’s like to serve abroad and be in a state of constant alertness.”In the interviews conducted midway through the intervention, the veterans emphasised some changes that had occurred in the way they perceived relationships. They all experienced a positive feeling of being part of the group of veterans during the NBT. Being surrounded by people with the same background, similar experiences, and challenges was considered more comforting than being with civilians.This understanding resulted in an atmosphere in the group in which they felt free to put memories and reflections into words. They expressed the importance of being in an ambience of understanding where no explanations were needed.In the same round of interviews as above, several of the veterans highlighted how the term ‘acceptance’ had become a cornerstone in their thinking about life. Gradually, the veterans expressed acceptance of what life might bring instead of fighting it. In addition, during the NBT, the veterans described different strategies they had developed to cope with stressful situations and quarrels with their families.“I need an instrument to control the turmoil and nerves in my body because I hate it. I hate having it […] it’s a heavy burden.”In the first interview, all the veterans related a history of long-term treatment with medication and/or psychotherapy. Some of the treatments had provided small improvements, but had also shattered hopes and dreams when the improvements did not last or even failed to materialise. Even so, in the first interview, all the participants expressed positive expectations to the intervention. However, their expectations differed and reflected their present life situation. Some felt the need for specific tools to help them to regain control:“You get anxious of this change [of oneself]. And where does it lead to? You see the weirdo on the street, and suddenly you imagine that’s the way you’re going.”Several of the veterans’ statements reflected their hopes and fears regarding their PTSD symptoms. Being disappointed could affect their mood and reinforce anxiety. For the veterans, the alternative to not getting better was frightening. One veteran with a background as a social worker said in the second interview:“Future? It’s almost like it’s just some letters on a piece of paper that I see. A mathematical formula on a white wall…”Another participant was unable to see a future for himself. He expressed it like this in the second interview, giving an impression of the deep worry that the veterans face:“Before, I was so preoccupied with saying, in six months I will have got so far with my disease. But I’ve dropped everything to do with time horizons and that sort of thing. Things must come as they are, the more I hurry, the more I stress myself, and the less energy I have to handle things.”However, at the end of the intervention, the veterans’ thoughts about the future had changed. Anxiety was considered less dominant. Instead, the veterans accepted the slow pace of the improvements they experienced, and time became an important factor in their healing processes. They expressed an awareness that they should not put pressure on themselves.“Now I manage to do some of those things that I had completely avoided before […] Yes I have a dream…being more independent [financially] through having a job. Sometimes I really wonder if I am that damaged that it’s a utopia for me…but, then I think, if I really could make it, I would create a life together with my son.”This expresses a growing insight into the condition among the veterans, and it acknowledges their own role in a recovery process. During the intervention and the year after the NBT, some of the veterans even experienced being able to handle their problems and imagine a future. A veteran expressed it like this in the final interview (one year after the intervention):The veterans described their identities as soldiers as strong and important parts of themselves in the first interview. Coming home, feeling different, and receiving a PTSD diagnosis had a huge impact on their lives. They said that being constrained and not being able to do the same things as before had resulted in their view of themselves as having changed, and this forced them to work on constructing another identity.“Then my boss comes up to me and grabs me. I remember, I tried to get away, but then he gave me a hug. And then it starts! A tiny little spark… And then the priest comes over, and he’s a fucking good priest. He’s like a dad. He always blesses us when we go on patrol, and it might look silly on television, but it’s bloody important when you’re in a place like that.”Almost all of the participants were young and had recently graduated when they were sent to war areas. They experienced how life as a soldier in a war zone often differed from their expectations, for example, that it was often difficult to distinguish enemies from civilians. The military rank system caused contrasting feelings: Some felt strengthened by rising up the ranks, while others felt the responsibility as a burden. There were also times when it was difficult to make situations fit into the rank system. One of the participants described a situation where he was accompanying a critically injured comrade into the helicopter to say goodbye. He felt this existential point between life and death where emotions exceeded the military rank within the group.“Well it’s a bit strange, but when I was in the military, I was walking in the night, it was much more comfortable if I had my rifle, although it was secured and only fired blanks…it was still my weapon, and it gave me some calmness and protection […] and even when I walk around as a civilian, I miss it, walking at night.”All the veterans expressed in the first and second interviews that fellowship, the uniforms, the weapons, and the common language of the military were important factors which bonded them together and gave them a common identity. One of them expressed (15 years after leaving the military) how he still felt strongly connected to his weapon:“I’m a stranger in the world, but also to my own body, and the only thing that ties me to the world is a sewing thread.”Some veterans described that after returning home and experiencing the changes as a result of PTSD, their self-perception gradually turned into a feeling of alienation. One felt like he was just an observer of his own life instead of actually living it:“a master in his own house.” This can be interpreted as PTSD had taken over the control of his life. However, one year later, in the last interview, when asked the same question, he answered:“I’ve found out that I don’t even have the foundations of the house…you build up a little, and find out that it must be in another way because it doesn’t work for me that way.”The NBT provided an insight into the participants’ self-perception. During the first interview, one veteran described how he was no longer “It’s a place where I feel respected, and they talk to me as a human being and not just a worker, so I want to give more, and I can push myself more […] I’ve got my professional pride back.”The participants said that when they were no longer able to serve as a soldier, finding a new occupation became an important task in constructing a new identity and being a respected member of society. When talking about his new job, one veteran said (interviewed one year after NBT):Being able to handle one’s own situation is a huge step in gaining control over life in spite of having PTSD.During the therapy, the veterans experienced changes in their way of thinking and handling life situations presented in the four above themes. ‘Lessons learned’ can be seen as the veterans’ own reflections on the whole process, from starting the NBT, to one year after it had ended.“So I try to do different things…there’s a dog’s playground next to my place, and for a period, I sat on a bench nearby. Partly not to be too close, but on the other hand, I wanted to be so close that I got some human contact without being in focus.”The veterans expressed how the NBT helped them take more initiatives  and find solutions (they could now use different tools) to the restrictions in their daily lives with PTSD.“One might think about things in a totally different way now, and it [the PTSD symptoms] makes you rise to the challenge, but at the same time, you become more handicapped…or what you might say.”One veteran reflected on how a better understanding and acceptance of himself could be a stepping stone to gaining more control of his life. However, it could also imply extra work:He used the word “handicapped”, which is usually associated with a physical disability. However, the term expressed his feeling of being unable to function on equal terms with others.”I’ve found out that everything does not have to be exactly in order all the time, right? Before, it meant a great deal to me that everything was in the right place. I think it’s because I found out that my head can’t deal with it.”Accepting one’s needs and capacities was part of the NBT. It was a challenge for some of the participants to implement acceptance in their daily lives, as this was considered to be in opposition to structure and order as an expression of being in control. One veteran described how he had felt that untidiness and things being out of place was unacceptable before he took part in the NBT. However, now this was seen as a positive thing:“I was not the only one who felt that way,—but we are soldiers and follow orders [..]”It takes time to implement some of the content from the NBT. During the third interview, one veteran talked about a frustrating episode in the garden: The horticultural therapist had asked all the veterans to hold hands in a circle and this was something he felt very uncomfortable doing. After a while, he spoke up and the therapist praised him for doing so:”I have changed now because I dare to say ‘no’, and it will be easier for me in my future life.”During the final interview, he mentioned the same episode again and said:First, a brief summary of the findings for each superordinate theme is presented. Then the results are discussed and related to the literature.In general, the veterans experienced changes in their lives with PTSD from baseline to one year after the intervention. In the first interview, they described their bodily experiences from having PTSD as pain, sleep disturbances, high arousal, and flashbacks. For some, the changes occurred in the intervention period, while others described these changes in the last interview one year after the intervention. Not only had the symptoms decreased, the veterans also expressed a feeling of control in relation to distress.The veterans described their relationships with their comrades during service as strong, and they felt guilty about leaving before completing their service or when comrades were killed. Being in a group of comrades that shared their experiences during the intervention helped them deal with the situation. Their relationships with family members were described as complex; the veterans wanted close relationships, but at the same time they were not always able to endure them. In the last interview, more veterans expressed how being in nature made them feel more calm, and how it was now less difficult to be with family and friends and to talk about their feelings.All the veterans expressed a fear that the condition might worsen due to earlier experiences of highs and lows in relation to treatment. In the beginning of the intervention, the veterans expressed it as almost impossible to imagine the future. This changed during the intervention period. Through NBT, they seemed to gain more control over their lives, and it brought them to a state in which they dared to imagine a positive future.The process of learning to accept the loss of their strong identity as a soldier and the creation of a new identity was described as demanding. For some veterans, being able to manage a job gave a feeling of being respected. During the intervention, the veterans improved their ability to find new solutions and strategies for handling challenges in their life in more appropriate ways.When comparing the participants’ bodily reactions, they were in line with frequently reported literature ,18,80,81The NBT offered the veterans an opportunity to join a group of fellow veterans. The veterans described how this fulfilled two of their needs: To be in a group representing the military culture with which they had lost connection, and to meet with people with similar experiences and PTSD symptoms.The importance of relationships with comrades during service has also been identified in other studies ,89, inclAll the veterans said that their families were very important parts of their lives, but at the same time the relationships were also a source of problems. During and after the NBT, the veterans handled their relationships with their families differently. Emotional numbing and the feeling of dissociation from their families were described as reasons for the complicated relationships. While numbness may be a useful emotion during war, it complicated the veterans’ family relationships. Emotional numbness is listed as one of the PTSD symptoms that affects both the veterans and their families ,26,94. AAll the veterans who participated in the NBT wanted their lives to be less affected by PTSD. They asked for tools to help them reduce bodily stress and understand their bodily signals. This could be interpreted as an instrumental way of viewing the body as a ‘thing’ one must gain control over. This view may have its roots in the military system with discipline and bodily control ,96, whicThe design of the study made it possible to follow the effect of the NBT through the veterans’ experiences. The theme “lessons learned” addressed the veterans’ reflections regarding the effect. They mentioned acting more appropriately in difficult situations and using tools from the NBT as some of the positive effects of the therapy. NBT helped the veterans to develop dynamic strategies, which had a positive effect on their relationships, identity, and dreams for the future. Working with mindfulness activities seemed to give a deeper insight into themselves and their resources. The veterans described how working with ‘acceptance’ as part of the mindfulness activities gave them the opportunity to develop and deal with a new identity; maybe different from who they were before, but no longer an inferior version. Mindfulness conducted in a nature environment may have been tolerated better by the veterans, because many of the sounds like birdsong and running water are well known to the brain, and because no sudden noises, for example honking horns and people shouting, occur.The NBT took place in nature and was an active contributor, both directly by simply being in nature, and indirectly through the horticultural activities. Kaplan  describeIn the NBT, the therapeutic acceptance of the veterans’ own experiences of their resources and support of their choice of activities  built up the veterans’ self-confidence. The veterans described how NBT influenced their ability to take the initiative and seek new solutions in their lives, and strengthened their capacity to construct a new identity. We do not necessarily see NBT as a treatment that is either a ‘stand-alone’ treatment or an ‘add-on’ therapy. The condition of the individual participant must be taken into account when determining the most beneficial treatment. Some of the veterans in this group had suffered from PTSD for just a few years, whereas as others had suffered from it for more than 18 years. Our results indicate that veterans who have been included in many ‘vet programmes’ or who have had bad experiences with medical treatment or who have felt that cognitive therapy was too provoking for their condition experienced the best results. This indicates that it is a suitable therapy for vulnerable veterans.The study has some limitations. The participants were recruited through advertisements in newspapers and social media and webpages for soldiers and veterans. Therefore, our sample may have been comprised by a group of especially motivated veterans or veterans with especially mild or severe symptoms. In addition, only male veterans responded. However, the veterans in our study differed with regard to age and time since the onset of PTSD. In addition, they all represented rather well-known PTSD symptoms. Therefore, we think that our results are transferable to other male veterans. We cannot know if the results also apply to female veterans. As the study is qualitative, the question of generalisability cannot be answered. Nevertheless, the participants in our study experienced an effect of the intervention and the effect was sustained after one year. This means that the intervention is experienced as effectual by some male veterans.The veterans of this study seemed to be split between two worlds. Living with the PTSD meant that at the beginning of the intervention, their awareness was in the military world; they had been enculturated to the military world with its extreme situations and they had lost the competences required to navigate in the civilian world. Their loyalty was also to the military world and was directed towards their comrades. They were locked emotionally in the traumatic situations they had experienced and brought their war identity home with them, where it did not fit in. They had to go through therapy to return home and adapt to a new civilian identity. The NBT helped them take on this new identity through a process where they, together with comrades, could gradually allow themselves to let go of their military identity. Even though they could not just take on their former identity again, the ability to relax again that they achieved through the experiences in nature helped them on their way to finding a new identity.After the ten-week NBT programme in a therapy forest garden, the veterans diagnosed with PTSD reported that their bodily symptoms were less burdensome, and that being in a group with people with a similar military background and culture had been beneficial. Moreover, they noticed an improved ability to take part in social activities with their families. They had learnt to be more accepting of their condition and the slow pace of recovery, including the need for developing new identities. This opened a door to imagining a future again with an ability to work and improved self-esteem. The veterans’ reflections on the NBT process at the follow-up revealed that tools from the therapy had enabled them to better handle potentially difficult situations and gain more control of their lives. This means that the results seem sustainable. In the last interview, all the veterans expressed that living with PTSD had become easier. Although the study is based on a small number of informants, the results seem promising and NBT should possibly be considered as a therapy complementary to the existing evidence-based therapies.


Background: The Head and Neck Cancer Working Group of Swiss Group for Clinical Cancer Research (SAKK) has investigated the level of consensus (LOC) and discrepancy in everyday practice of diagnosis and treatment in head and neck cancer.Materials and Methods: An online survey was iteratively generated with 10 Swiss university and teaching hospitals. LOC below 50% was defined as no agreement, while higher LOC were arbitrarily categorized as low (51–74%), moderate (75–84%), and high (≥85%).Results: Any LOC was achieved in 62% of topics (n = 60). High, moderate, and low LOC were found in 18, 20, and 23%, respectively. Regarding Head and Neck Surgery, Radiation Oncology, Medical Oncology, and biomarkers, LOC was achieved in 50, 57, 83, and 43%, respectively.Conclusions: Consensus on clinical topics is rather low for surgeons and radiation oncologists. The questions discussed might highlight discrepancies, stimulate standardization of practice, and prioritize topics for future clinical research. This is the fourth part of the article “A Review of Controversial Issues in the Management of Head and Neck Cancer: A Swiss Multidisciplinary and Multi-Institutional Patterns of Care Study,” providing the results for the items concerning biomarkers, each followed by a short discussion if deemed relevant. The details of the methodology is presented in the first part of this series.Imaging findings indicating hypoxia and/or central necrosis are not standard factors influencing the treatment decision: no consensus.➢ In 5/10 centers, imaging findings indicating hypoxia or central necrosis affects the decision for the primary treatment modality.According to the literature , tumor hIn tumors with presence of diffuse hypoxia a systemic approach using a hypoxic cell cytotoxin or anti-growth factor drugs might be beneficial to overcome the limitations of hypoxia . AlternaAnyhow, regarding the limited evidence for the role of imaging findings indicating hypoxia, it is quite remarkable that half of the centers in Switzerland integrate them in treatment decisions.De-differentiation grade is not a standard factor influencing the treatment decision: no consensus.➢ De-differentiation grade of tumors also influences treatment decision in 5/10 centers. This question did not differ between squamous cell cancer and other malignancies of the head and neck.In salivary gland carcinoma, the histologic grade is a significant predictor of treatment response and an established factor for therapeutic decisions, but due to the rarity and wide variety of different tumor types the definition of predictive grading schemes is challenging .In HNSCC, histologic grade is not part of the current staging criteria, probably because its prognostic impact remains controversial. Weijers et al.  found noDetermining the HPV status is a standard practice in oropharyngeal squamous cell carcinoma (OPSCC): high LOC (100%).➢ There is no standard method established for the definition of HPV status: no consensus.➢ All (10/10) centers regularly determine the HPV status in OPSCC. The definition of an HPV attributable tumor is IHC overexpression of p16 as a single marker (5 centers), HPV high-risk type DNA positivity by polymerase chain reaction (PCR) (2 centers); HPV high risk type DNA positivity by ISH (1 center) and p16, followed by PCR if needed according to College of American Pathologists guidelines (1 center).The survey was performed prior to the release of the 8th edition of the UICC TNM classification system, implementing p16 IHC as a crucial biomarker for staging of OPSCC. Nevertheless, all centers had already started to routinely determine the HPV-status in OPSCC. Interestingly, the definition of a positive HPV-status widely differs between the centers, reflecting the lack of a worldwide-accepted consensus for the accurate definition of an HPV-driven cancer. In the new UICC staging system (8th edition), p16 is accepted for practical and cost-related reasons considering the guideline to be international , but theINK4A (inhibitor of cyclin-dependent kinase 4) overexpression in tumor tissue by IHC is used as a surrogate marker for HPV-driven HNSCC : low LOC (60%).➢ In 6/10 centers, HPV status is routinely determined in lymph node metastases without evidence of a primary tumor.18FDG-PET/CT is adversely affected by false positives from hypermetabolic oropharyngeal lymphoid tissue .➢ HPV status does not influence the treatment decision in 8/10 centers. Two centers stated that they may consider changing the treatment intensity.Although HPV-positivity in OPSCC is an established positive prognostic marker, treatment decisions should so far not be influenced by it. There is a lot of ongoing discussion about treatment de-escalation in this low-risk tumor but centers should wait for the shortcoming results of prospective clinical trials to decide on less intensive treatment regimen.In summary, there is no consensus regarding the applicability of imaging findings indicating hypoxia as well as histological differentiation grade. In all centers, the determination of the HPV-status is a standard practice in oropharyngeal squamous cell carcinoma rather than in cancer of unknown primary. Since the survey was performed prior to the release of the 8th edition of the UICC TNM classification system it is interesting to see, that at that time the importance of HPV infection especially in CUP was not evident in almost half of the centers in Switzerland.Furthermore, there is a lack of standard method established for the definition of HPV-status ranging from p16 IHC as a single marker, HPV-DNA by PCR or ISH as single markers or the combination of both reflecting the lack of a worldwide-accepted consensus for the accurate definition of an HPV-driven cancer. In the majority of centers, there is no therapeutic consequence of HPV-testing in both OPSCC and CUP due to lack of practice guidelines based on prospective clinical trials.The findings of our survey indicate a low LOC among head and neck oncologists working in academic and multidisciplinary setting in 10 Swiss institutions. The highest LOC was achieved among medical oncologists, whereas the lowest was observed among head and neck surgeons. On the other hand, this level of disagreement may also depend on the topics chosen for the survey, and not necessarily the heterogeneity within the disciplines. It is also interesting to witness a low LOC regarding topics, where a high level of evidence actually does exist, and vice versa. This article is expected to serve the head and neck oncologists to be aware of their discrepancies and to stimulate discussion toward standardization of practice and prioritize topics of future clinical research.All datasets generated for this study are included in the manuscript/supplementary files.GH, MB, OE, PD, and PP: conception and design. OE and PP: collection of data. Generation of the initial and final versions of the questions, drafting of the manuscript, and approval of the final version by all co-authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Invasive cervical cancer is the most prevalent cancer among women in Sub-Saharan Africa. In 2013, the World Health Organization (WHO) emitted recommendations to start Highly Active Antiretroviral Therapy (HAART) regardless of CD4 count. Although HAART has been shown to reduce the prevalence of high-risk human papillomavirus (HR-HPV) genotypes, it is unclear whether it confers a protective effect specifically for HPV 16. This review summarizes the existing evidence regarding the effect of HAART on HPV 16 infection, as this genotype may not be influenced by immunity level and explores its implications for Sub Saharan Africa. A comprehensive literature review was undertaken and quality assessment was carried out on the selected papers. Four cohort studies and three cross-sectional studies were identified for which the overall quality score assessment ranged from weak/moderate (Score of 1.8) to strong (Score of 3). The evidence yielded by our review was conflicting. Thus, the high heterogeneity between study populations and results did not allow us to draw any firm conclusions as to whether HAART has an impact on HPV 16 acquisition/prevalence. As only three studies were conducted in Africa, there are insufficient grounds for solid comparison between geographic regions. In light of inadequate data, HPV unvaccinated women on HAART should still receive more frequent follow-up. In 2013, an estimated 35 million people were living with HIV worldwide .Human papillomavirus (HPV) is a sexually transmitted infection and high-risk (HR) HPV DNA has been shown to be present in 99.7% of cervical cancers worldwide . FurtherAmong the HR-HPV genotypes, HPV 16 and HPV 18 have the greatest oncogenic potential accounting for about 70% of all ICC .Prophylactic HPV vaccines are likely to reduce the future burden of cervical cancer to a significant extent, particularly where screening is scarce, such as in Sub-Saharan Africa. The primary target group in most countries recommending HPV vaccination are adolescent girls aged 9–14. Prompted by the rapid effectiveness seen in many industrialized countries where the age range for HPV vaccination has been extended to cover women up to the age of 26, in 2016 the WHO revised its position and is hence recommending vaccination for this age group in resource-poor settings in order to step up HPV vaccine uptake. This, in turn, is expected to yield benefits at community level .Furthermore, access to HAART in sub-Saharan Africa has greatly improved over the past decade, increasing life expectancy for women living with HIV . In sub-2 = 51%) [A recent systematic review suggests that duration of HAART along with the CD4 count may reduce the prevalence of HR-HPV in sub-Saharan Africa . A recen2 = 51%) . This isWhilst HPV genotypes other than HPV 16 are often better controlled by host’s immune responses, it is hypothesized that HPV 16 is better equipped to evade immune surveillance , 19. SomThe primary objective of this review is to elucidate the impact and effects of HAART on prevalence/incidence of HPV 16 compared to other genotypes. The second objective is to highlight knowledge gaps for further research and specifically how they relate to the sub-Saharan African context.We conducted this systematic review based on a pre-defined search protocol that conformed to the criteria set out by the PRISMA statement. PubMed, CINAHL, Global Health, Scopus, and EMBASE databases were searched between January 1997 and March 9, 2018 .The domains of the search terms were HPV 16 OR human papillomavirus genotype 16 AND acquisition OR incidence/detection OR prevalence AND Anti-HIV Agents OR Antiretroviral Therapy OR High Active OR antiretroviral OR ART OR HAART We considered the following four components  to assess and categorize studies to be included in this review. The population involves HIV-positive women with HPV 16 infection (among other genotypes). The intervention examined in this study is the administration of HAART, defined as at least 3 antiretroviral drugs belonging to 2 different drug classes (Non-nucleoside reverse transcriptase inhibitors or nucleoside reverse transcriptase inhibitors or Protease Inhibitors) and, if possible, the duration of treatment. Women on HAART for at least 6 months are being compared to women not receiving HAART or just starting HAART. The outcomes of interest are: Prevalence or incidence or acquisition of HPV 16 for the two comparison groups.We initially intended to conduct a systematic review and meta-analysis to answer the question on whether HIV positive women on HAART have higher probability to prevent HPV 16 infection than HIV-positive non-users of HAART. However, due to the scarcity and methodological heterogeneity of retrieved studies, and the lack of similarity of measure of effect of the various studies (Odds ratio vs Hazard ratio), pooling estimates would be inappropriate. We therefore re-oriented this study to a systematic review, without conducting a meta-analysis. The search strategy was designed by a medical librarian to identify studies reporting HPV 16 acquisition in women on HAART. We conducted this review on a pre-defined search protocol that conformed to the criteria set out by the PRISMA  statemenAll studies were independently reviewed and critically evaluated for inclusion by three authors . All data were extracted independently and in duplicate manner by the three investigators . Possible cases of disagreement were resolved on an individual basis via detailed discussion among the authors of the justification for exclusion/inclusion. The following items were recorded: first author, publication year, study design, type of sampling, sample collection method, study population, total sample size, HIV status, incidence/prevalence of HPV 16 and duration of HAART use, baseline of CD4 count in study population, sexual behavior, prevalence of multiple HPV genotypes, past history of STI and if the analysis included adjusting for confounding factors. The adjusted Odds ratio (a)OR, adjusted Hazard Ratio(a)HR and 95% Confidence interval (CI) were recorded and if the 95% CI did not include 1 and no p- value was given, it was considered significant.Because of the lack of randomized trials retrieved , we considered prospective cohort/longitudinal studies to be of highest quality. We reported an overall score (from 1 to 3) based on our assessment of the overall quality of evidence for the following parameters:Minimization of selection bias: (Score “3” = strong), the participants were likely to represent the target population; (Score “2” = moderate), somewhat likely; (Score “1” = weak), not likely to represent the target population.Study design: (Score “3” = strong), a prospective study with a large sample size (> 200) ; (Score Confounders: (Score “3” = strong), controlled for all relevant confounders; (Score “2” = moderate), controlled for some confounders; (Score “1” = weak), control for confounders not specified.Withdrawals, loss to follow-up and dropouts: (Score “3” = strong), description of a strategy to minimise the loss to follow-up, reported withdrawals and drop-outs (80–100% of participants completed the study) ; .On March 8, 2018, we retrieved 210 articles from PUBMED, CINAHL, Global Health, Scopus and EMBASE, of which 103 duplicate titles were identified. After screening the 107 remaining titles and abstracts, 11 were full text-screened and 7 were eventually included in this review. No disagreement between the three reviewers emerged on whether to include or exclude any given study.Studies varied in reporting, baseline of CD4 count in study population, sexual behavior, prevalence of multiple HPV genotypes, and past history of STI .A total of 3298 HIV infected women were included in the studies retrieved for this review. Sample sizes for these studies ranged from 225 to 1010 HIV infected women.Seven studies were eventually included in this review. These studies varied by their design including prospective (n = 4) and retrospective cohort (n = 1) and cross-sectional (n = 2) studies. Four studies were conducted in developing countries (3 in sub-Saharan African countries and 1 in India) while the other three were in developed countries . Table 2The overall quality of evidence pertaining to the association/impact of HAART on HPV 16 prevalence/incidence ranged from weak/moderate (Score of 1.8) to strong (Score of 3). One study had a score of 1.8 , 29 fourAll seven studies explored the impact of HAART on HPV 16 prevalence or incidence, of which four supported our hypothesis that HAART has no impact on reducing HPV 16 acquisition or prevalence. In a longitudinal cohort study of 750 HIV-infected women, Blitz   found thIn a prospective cohort study on adolescents,   HAART diWe identified three studies, which refute our hypothesis. Zeier  , in the The findings from this review are conflicting and contradictory. Therefore, a conclusive answer on the impact and effect of HAART on the incidence and prevalence of HPV 16 is not possible. The overall quality of evidence pertaining to the association/impact of HAART on HPV 16 prevalence/incidence ranged from weak/moderate to strong, which further reduces our capacity to explore the association.Our study builds on the recent global meta-analysis , which eMoreover, given the fact that only three studies took place in Sub Saharan Africa, we are limited in our capability to compare findings between this geographical region and others. This was exacerbated by the high degree of heterogeneity in quality, reporting, setting, study population and results. The baseline CD4 count was mostly given upon enrollment of in the study, but not when HAART was initiated. And in only one study , the medFurthermore, only one study examined past history of STIs . A meta-Moreover, only one study  examinedThe major strength of this study is that there has been a thorough attempt to search five databases and retrieve all the relevant existing literature. Nonetheless, this study does have limitations as well, including our incapacity to carry out a meta-analysis due to the heterogeneity of study designs and their measurement of effect.In light of the inconclusive findings of the review, unvaccinated HIV-infected women may still require more regular monitoring than once every three years if tested negative by VIA or cytology, as recommended by the WHO for this population , despiteA major gap is to elucidate the HPV 16 Basic Reproductive Number (R0) in various types of populations, which may reveal what percentage of the population needs to be vaccinated. In light of the rollout of HAART at a higher CD4 count in sub-Saharan Africa, it is imperative that the prevalence of HPV 16 in HIV-infected women per sub-Saharan country be better established. Knowledge about the specific HPV type distribution in the HAART era is crucial to guide HPV-based screening intervals and the feasibility of catch-up campaigns focusing on older HIV-infected girls.Recent literature suggests that the lesser impact expected of the HPV vaccine in older HIV-infected girls, due to a potential higher risk of prior exposure to vaccine-targeted genotypes, may be compensated by the potential of the HPV vaccine to prevent recurrence of CIN 2–3 among women treated for HSIL . In thisIn light of the increasing number of HIV-infected women accessing HAART at a less immunocompromised state, the impact of different levels of immune reconstitution conferred by HAART on HPV 16 acquisition as a stand-alone or as co-infection with other HR—HPV genotypes should be elucidated. Hence, it is imperative that more studies on the sub-Saharan continent be undertaken and disaggregated baseline data be collected by HPV 16 status in order to adjust for potential confounders.As the prevalence of BV in sub-Saharan women is among highest in the world , it is pRollout of HAART in sub Saharan Africa has brought forth concomitant increases in drug resistance rates, which can be attributed to the empirical first and second line antiretroviral regimens and the clinical or immunological definitions of treatment failure without plasma viral load monitoring . Data onOur limited data does not enable us to yield conclusive findings. To err on the side of caution, our data suggests that unvaccinated women on HAART may require more frequent follow-up. The scarcity of data on acquisition of specific HPV genotypes by HAART regimens is reflective of the lack of data found on the immuno-epidemiology of oncogenic genotypes and their synergistic interactions prior and after initiation of HAART. Apart from recent literature suggesting a protective effect of HAART against HR—HPV infection, further research on the impact of HAART on HPV 16 infection is needed with a view to designing an optimal screening and vaccine strategy in HIV-infected women in Sub Saharan Africa.S1 File(TIFF)Click here for additional data file.S2 File(TIFF)Click here for additional data file.S3 File(TIFF)Click here for additional data file.


Genotyping HPV from samples tested positive to careHPV™ assay in rural and remote areas of Brazilian territory.A total of 5079 women were enrolled in an opportunistic screening from the Barretos Cancer Hospital, through mobile units or ambulatory unit. All careHPV™ hr-HPV positive samples were tested by a Luminex-based protocol in order to evaluate the HPV infecting types.Positive hr-HPV results were obtained in 10.6% (536/5068) of women. Among these cases, HPV-56 and HPV-51 were the most common types detected in 32.3% and 31.4%, respectively. HPV-53 (20.5%), HPV-18 (18.5%), HPV-58 (17.6%), HPV-52 (16.0%) and HPV-16.6%) were the other most frequent types detected. These frequencies represent prevalences of 2.35%, 2.12%, 2.02%, 1.84% and 1.80% respectively, within the population studied. Regarding low-risk HPVs, HPV-6 was detected in 12.9% of the samples. The less frequent types (<3%) were: HPV-70, HPV-11 and HPV-26.The most frequent types detected were: HPV-56, HPV-51, HPV-53, HPV-18, HPV-58, HPV-52 and HPV-16 according to decreasing rates. This is particularly disturbing if considers that the majority of these cases are found in developing and poor countries of Africa and South America ,3.HPV vaccination is a well-tailored option for primary prevention tool of HPV-related cancer. Currently, a number of evidences strongly suggest that HPV vaccination is an important implement to reduce the prevalence of malignancies induced by persistent HPV oncogenic types infection in women underprivileged of medical assistance. Recently, an elegant report conducted by Hall and colleagues indicates the possibility of cervical cancer eradication within the next 20 years in Australia by the high-coverage vaccination and screening. The aim is to decrease the rates to four new cases per 100,000 women per year, to consider cervical cancer as an extinguished problem of public health in Australia . Arbyn aHowever, despite to the scientific encouragement towards vaccine implementation, there are many restrictive elements for HPV vaccines application with evident reluctance in many settings of the general population, which represent a serious obstacle for HPV-induced lesions reduction, which must be overcomes to increase the vaccine coverage that comprise psychological, religious and/or cultural reactions .The report of Muñoz and colleagues defined with robust data from different countries the most prevalent HPV types worldwide . This isConsidering all these facts, we sought to investigate specific HPV types prevalence in rural and remote areas of Brazilian territory in order to draw a map of HPV types distribution in a country with continental dimension.2This was a retrospective evaluation of individual HPV types prevalence performed within a casuistic planned for a prospective study carried out from March to December 2010 of women attended at the Ambulatory of Prevention Department and the mobile units of Barretos Cancer Hospital (BCH), which assisted the Brazilian rural and remote areas, and these samples were previously underwent to Surepath Liquid-based Papanicolaou, and the HPV detection was performed using the careHPV™ molecular test .The project was approved by the Research Ethics Committee of the Barretos Cancer Hospital - Pio XII Foundation, protocol number 404/2010.2.1A total of 3079 volunteer women aged over than 18 were invited to participate in a opportunistic screening available at the mobile units of the Barretos Cancer Hospital, which assisted the states of Goiás, Minas Gerais, Mato Grosso and São Paulo. At this stage, there was no randomization of the studied group and the samples were collected by health professionals (careHPV™ test and Liquid-based Papanicolaou test).2.1.1in loco; the remaining samples were tested at the Research Molecular Center of BCH.The samples collected to careHPV ™ medium were initially analyzed in the mobile unit in ten out 63 cities visited by the unit: Mineiros (GO), Portelândia (GO), Alto Araguaia (MT), Alto Garças (MT), Alto Taquari (MT), Rondonópolis (MT), Pedra Preta (MT), Guiratinga (MT), São José do Povo (MT), Itiquira (MT); totalizing over than 400 samples analyzed 2.2Cervico-vaginal samples tested for careHPV™  were maintained under at 15 °C to 30 °C for two weeks, and/or 2 °C to 8 °C for four weeks or −20 °C for two months. Those samples that were tested positive in the careHPV test were maintained 2 °C to 8 °C.2.3DNA from the positive samples maintained on the careHPV™ medium were extracted using the QIAmp MinElute Virus Vaccum kit (Qiagen Co) according to the manufacturer's protocol. Extracted DNA were kept in at −20 °C.2.4HPV positive samples using the careHPV™ test were further genotyped using the Luminex methodology . This methodology uses oligonucleotide probes coupled polystyrene beads containing two different fluorophores inside that allows the simultaneous detection of at most 100 different specific HPV types. In this study, probes used allowed the identifications of 21 viral types: i) low risk: 6, 11 and 70, ii) high risk: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82. PCR reactions were performed using the Qiagen Multiplex PCR kit  and a mixture of specific biotinylated primers capable of amplifying a fragment of the E7 gene, followed by bead-based Luminex technology . Primers for the amplification of β-globin were also added to evaluate the quality of template DNA. For each probe, MFI (mean fluorescence intensity) values obtained when no PCR product was added to the hybridization mixture was considered the background values. Cutoffs were computed by adding 20 MFI to 1.1X the median background value. Whenever a positive signal for HPV and β-globin was not obtained, the sample was considered inadequate. Those cases in which the positive signal for HPV was not observed, but β-globin was detected, samples were considered negative as well.2.5The prevalences of HPV specific types were computed. Statistical analysis was performed using IBM® SPSS® Statistics  version 20.1 for Windows  and MedCalc® version 11.1 for Windows .3Women tested positive for HPV (10.0% or n=307) using the careHPV test showed mean age of 41.0 years. The socio-demographic findings achieved previously did not show any statistically significant association between HPV positivity and tobacco use, number of parities, number of sexual partners and contraceptive [3.1From 443 samples available for analysis, high-risk HPV was detected in 369 samples, 72 were positive for high and low risk HPV simultaneously and 2 cases tested positive for low risk HPV (both HPV6).Single HPVs infections were observed in 25.3% (112/443) of the samples, two of them by low-risk HPV (HPV 6). Conversely, 74.7% (331/443) of the samples had multiple infections solely with high-risk types or with high- and low-risk HPVs.HPV-56 and HPV-51 were the most common types, and were detected in 32.3% and 31.4% of the samples, respectively. This represents 3.7% of HPV prevalence, considering the percentage of HPV positive women found in this study (11.5%). Following HPV-53 (20.5%), HPV-18 (18.5%), HPV-58 (17.6%), HPV-52 (16.0%) and HPV-16 (6%) were the most frequent, which represents a prevalence of 2.35%, 2.12%, 2.02%, 1.84% and 1.80%, respectively, according to the percentage of HPV positive women studied. Regarding low-risk HPVs, HPV-6 type was detected in about 12.9% of the samples. The less frequent detected types (<3%) were: HPV-70, HPV-11 and HPV-26. 4The general positivity for high-risk HPV reported in this current study was 10.6%: 11.5% observed in the casuistic of cases collected at the Prevention Department of HCB and 10% detected in women examined at mobile units, respectively. Data documented by other Brazilian studies showed positive rates ranging from 9.7% to 10.5% . RecentlHPV genotyping performed within the positive samples showed in descending order, higher frequencies of the HPV-53, HPV-18, HPV-58, HPV-52, and HPV-5 and HPV- HPV-16, with frequencies of detection of 32.3%, 31.4%, 20.5%, 18.5%, 17.6%, 16.0% and 15.6%, respectively. Moreover, the overall HPV typing demonstrated that HPV types frequencies is particularly different if compared to the frequencies reported in literature, once HPV56, HPV51 and HPV53 were significantly more prevalent than HPV 16 and HPV18, presumed as the more prevalent HPV types worldwide . In a reIn addition, it is necessary to note that differences in the methodologies used to detect and type HPVs can certainly influence data of the different studies regarding HPV types prevalence.Finally, the importance of HPV typing seems to be closed related to the contribution that these observations can provide to the strategies of vaccination in different regions of the world. This issue is not negligible if one takes in account that HPV vaccination should be planned for certain regions of the world, considering that the variation of HPV typing could infer a bias of HPV vaccine efficacy if the most prevalent types are not included among types selected for bivalent, quadrivalent or nonavalent vaccine currently available in the medical armamentarium. Cross-protection is presently and potentially expectable for all of the 3 licensed HPV vaccines provide protection against HPV types not included in the vaccines. It is well-stablished that bivalent and quadrivalent HPV vaccines provide some level of cross-protection against high-risk HPV types: 31, 33 and 45 HPV types. However, the degree of cross-protection against HPV types of nonavalent vaccine is not yet documented .In conclusion, our study demonstrated that HPV types have a significative variation prevalence in Brazilian territory; data can be improved with robust increasing casuistry.The careHPV ™ test kits, as well as equipment, preservation media, consumables and brushes for molecular testing, were provided by Qiagen® Brazil.National Institute of Research and Technology in HPV (INCT - HPV) of São Paulo, through the CNPq process nº 573799/2008-3, FAPESP process nº 2008/57889-1, through and CNPq process nº 203348/2014-1.The study was supported financially (grant-aid) of the


In the article titled “The Impact of Type 2 Diabetes Mellitus on Long-Term Prognosis in Patients of Different Ages with Myocardial Infarction” , the aut


Conditional keratinocyte-specific p38δ ablation (p38δ-cKO∆K) did not influence the latency, incidence, or multiplicity of chemically-induced skin tumors, but led to increased tumor volume in females during the TPA promotion stage, and reduced malignant progression in males and females relative to their wild-type counterparts. In contrast, conditional myeloid cell-specific p38δ deletion (p38δ-cKO∆M) inhibited DMBA/TPA-induced skin tumorigenesis in male but not female mice. Thus, tumor onset was delayed, and tumor incidence, multiplicity, and volume were reduced in p38δ-cKO∆M males compared with control wild-type males. Moreover, the percentage of male mice with malignant tumors was decreased in the p38δ-cKO∆M group relative to their wild-type counterparts. Collectively, these results reveal that cell-specific p38δ targeting modifies susceptibility to chemical skin carcinogenesis in a context-, stage-, and sex-specific manner.Activation and/or upregulated expression of p38δ are demonstrated in human skin malignancies including cutaneous squamous cell carcinoma, suggesting a role for p38δ in skin carcinogenesis. We previously reported that mice with germline deletion of the p38δ gene are significantly protected from chemical skin carcinogenesis. Here, we investigated the effects of cell-selective targeted ablation of p38δ in keratinocytes and in immune (myeloid) cells on skin tumor development in a two-stage 7,12-dimethylbenz( RAS genes are widespread in human cancers, including CSCCs [2-terminal kinases , and p38 MAPKs  [Cutaneous squamous cell carcinoma (CSCC) is the second most common type of skin cancer, with an estimated 700,000 or more patients diagnosed annually in the United States . The incng CSCCs . Mitogenng CSCCs . The MAPnd p38δ) ,5,6,7. TThe p38δ isoform is abundantly expressed in cutaneous epithelia, and is required for appropriate cell proliferation and differentiation in human keratinocyte monolayer and organotypic culture models ,10. HoweHA-transformed keratinocytes following orthotopic grafting onto nude mice by inducing transcriptional changes linked to tumor suppression [HA-induced tumorigenesis in a cell-autonomous manner. Furthermore, systemic p38δ loss heightened the initial inflammatory response in pre-neoplastic murine skin following a short-term DMBA/TPA challenge [Our laboratory previously reported that mice with systemic (germline) deletion of p38δ were resistant to chemically-induced skin tumorigenesis and to oncogenic K-ras-driven lung tumorigenesis, indicating that p38δ promotes tumor development in vivo . The esspression . These fhallenge . The corhallenge ,23,24,25hallenge ,26. Systhallenge .p38δ is expressed not only in epithelial cells, but also in immune, endothelial, and mesenchymal cells; reciprocal communications between these cells and incipient tumor cells have been shown to regulate tumor development and progression. Therefore, the functional involvement of non-epithelial cell-derived p38δ in skin tumorigenesis cannot be excluded. Notably, hematopoietic cell p38γ and p38δ were shown to be the main contributors to colitis-associated tumor initiation in a colorectal cancer mouse model . ∆K) or immune (myeloid) cells (p38δ-cKO∆M). Cell type-specific loss of p38δ revealed stage- and sex-dependent effects of p38δ inhibition on skin carcinogenesis in vivo, suggesting differential mechanisms of epithelial and myeloid cell p38δ in the regulation of skin tumor development. In the present study, we utilized conditional p38δ knockout mice to investigate skin tumor development in response to a two-stage DMBA/TPA chemical skin carcinogenesis protocol. In these mutant mice, genetic ablation of p38δ expression was targeted to keratinocytes . We observed efficient p38δ ablation in keratinocytes, while the levels of p38δ expression in heart and liver remained unchanged, indicating that the p38δ ablation was keratinocyte-specific , the incidence of tumors in p38δ-cKO∆K mice was significantly reduced during the malignant progression stage of the DMBA/TPA regimen . In addition, total and malignant tumor multiplicities were significantly reduced in p38δ-cKO∆K female mice compared to WT females, and benign tumor multiplicity was significantly lower in mutant female mice than mutant male mice . The efficiency of the deletion of p38δ in myeloid cells using this approach was previously confirmed [ΔM newborn and adult mice was histologically indistinguishable from that of WT control littermates (data not shown). We then subjected cohorts of WT and p38δ-cKOΔM male and female mice to the DMBA/TPA skin carcinogenesis regimen described in the Materials and Methods and outlined in Myeloid cells, including macrophages, neutrophils, monocytes, dendritic cells, and granulocytes, are recruited to the tumor microenvironment to regulate tumor growth and progression . onfirmed . Consistonfirmed , the epiΔM mice compared to WT males , leukocytes (CD45+), and T cells (CD3+) in the peritumoral stroma of SCCs from p38δ-cKOΔM male mice compared to WT male mice  genetic deletion of p38δ inhibited DMBA/TPA-induced skin tumorigenesis via attenuation of the proliferative ERK1/2-AP1 pathway and suppressed K-Ras-driven lung tumorigenesis, indicating a tumor-promoting function for p38δ in skin and lung tissue in vivo . Others ΔK mice exhibited significantly reduced overall and malignant tumor incidences compared to WT mice at the end of the carcinogenesis experiment (51 weeks post-initiation with DMBA), indicating that keratinocyte-intrinsic p38δ functionally contributes to the malignant progression of DMBA/TPA-induced skin tumors. Our present studies revealed that, unlike systemic p38δ ablation, conditional ablation of p38δ in epidermal keratinocytes did not protect mice from skin tumor development during the initial stages of the DMBA/TPA regimen, suggesting that keratinocyte-intrinsic p38δ function is not necessary for the initiation and promotion stages of DMBA/TPA skin tumor development. In contrast, during the malignant progression stage of the DMBA/TPA regimen, p38δ-cKOΔK mice, in which p38α is hyper-activated upon keratinocyte p38δ loss in response to TPA-induced pro-inflammatory stimulation [ΔK female animals showed significantly lower total and malignant tumor multiplicities than their WT counterparts at the end of the carcinogenesis experiment -dependent signaling mechanism, given that the p38α isoform is hyper-activated in initiated keratinocytes and incipient tumor cells in the absence of p38δ , and thamulation . This hyperiment , underscΔM male mice had a reduced incidence of malignant tumors, suggesting that myeloid cell-intrinsic p38δ is essential for malignant progression of DMBA/TPA-induced skin tumors in males. These findings support the notion that tumor-extrinsic myeloid cell-derived p38δ functionally contributes to chemically-induced skin tumor development in male mice. Furthermore, using immune (myeloid) cell-specific p38δ targeting, we demonstrated that male, but not female, mice with myeloid p38δ loss were significantly protected from DMBA/TPA-induced skin tumor development during both the promotion and malignant progression stages of the DMBA/TPA regimen, and exhibited an increased tumor latency, as well as reduced tumor incidence, multiplicity, and volume, compared to WT male littermates. In addition, mutant p38δ-cKOH2 or anti-tumorigenic immune-stimulatory TH1 states, respectively [H2-driven myeloid cells could be reprogrammed to instead promote anti-tumor immunity has been tested in several tissue-specific cancer models (reviewed in [+), leukocytes (CD45+), and T cells (CD3+) seen in our data correlated with reduced SCC tumor size and reduced tumor cell proliferation in p38δ-cKOΔM male mice compared to their WT counterparts (HA-initiated mouse keratinocytes [TGFBR2) and Fcγ receptors (FCGR2B and FCGR3). Given that TGFβ has been implicated in induction of pro-tumorigenic M2 macrophage and N2 neutrophil polarization, while inhibition of TGFβ signaling resulted in switching to an anti-tumorigenic N1 phenotype , and thiewed in , it is cΔK and p38δ-cKOΔM mice, supporting the significance of the sex-specific role p38δ signaling plays in both the tumor parenchyma and myeloid component of the tumor stroma. An unexpected aspect of our findings was uncovering the sex-dependent differences in specific features of the DMBA/TPA chemical skin carcinogenesis process in both p38δ-cKOIn summary, by focusing on functional outcomes of cell type-specific p38δ genetic targeting in an experimental model of chemically-induced mouse skin carcinogenesis, our findings support that p38δ has essential in vivo context-, stage-, and sex-dependent roles in cutaneous carcinogenesis, and contributes both cell-autonomous and paracrine effects during skin tumor formation. Overall, along with work by other groups, our data suggest that there are potential therapeutic benefits of targeting p38δ for the treatment of cancer. Furthermore, our data provide a rationale to validate an innovative approach of cell-specific targeting of p38δ for the treatment of cancer in a sex-optimized manner. The regulatory mechanisms underlying sex disparities in skin carcinogenesis have yet to be elucidated; our mouse models with cell-specific p38δ ablation will be instrumental in addressing these disparities at the molecular level in an in vivo setting.DMBA, TPA, bromodeoxyuridine (BrdU), Hoechst 33342, and p38α antibody were purchased from Sigma . For immunofluorescence experiments, we used p38δ antibody obtained from Santa Cruz Biotechnology . BrdU antibody was from Chemicon . For immunoblot experiments, we used p38δ antibody purchased from the Division of Signal Transduction Therapy  . AntibodloxP-flanked alleles of Mapk13, the gene encoding p38δ, (p38δflox/flox), were generously supplied by Professor Romeo Ricci, with permission from Boehringer Ingelheim Pharmaceuticals Inc. . The p38δ floxed allele was generated by homologous recombination in embryonic stem cells in which the first exon (containing ATG) was flanked by two LoxP sites. The detailed description of the targeting strategy was provided by Sumara et al. [flox/flox mice were backcrossed to a C57BL/6 genetic background for at least six generations. Mice with keratinocyte- and myeloid cell-specific ablation of p38δ were generated by crossing p38δflox/flox mice with Keratin 14 (K14)-Cre [ΔK  and p38δ-cKOΔM  were compared to their wild-type control littermates (p38δflox/flox and p38δflox/+) by their response to the two-stage DMBA/TPA chemical carcinogenesis regimen.All animal studies were approved by the Washington University School of Medicine (WUSM) Animal Studies Committee . Mice were housed under pathogen-free conditions and handled in accordance with National Institutes of Health guidelines. Mice with a et al. . p38δfloK14)-Cre  and LysoK14)-Cre , respectGroups of 8-week-old male and female mice were subjected to the DMBA/TPA tumor induction regimen as previously detailed . BrieflyΔK male and female littermates two days after shaving. Starting 5 days post-DMBA or vehicle treatment, mice were treated every other day with four topical applications of TPA (12.5 μg in 200 μL acetone per mouse per each dose) or acetone vehicle. Total skin lysates were isolated from full-thickness dorsal skin 2 h after the final TPA application.A short-term topical DMBA/TPA treatment was carried out as previously detailed . Brieflyt-test  to determine statistically significant differences.Full-thickness dorsal skin samples were collected, protein lysates were prepared, protein concentrations of the lysates were determined, and immunoblot analysis was performed as previously detailed ,18. IL-1Dorsal skin or tumor samples were fixed in 4% paraformaldehyde in PBS, dehydrated in ethanol, embedded in paraffin, and sectioned at 5 μm. For immunofluorescence detection, sections were deparaffinized, rehydrated, and subjected to antigen retrieval by microwaving in 10 mM citrate buffer (pH 6.0). After blocking, sections were stained with primary antibody at 4 °C overnight in a humid chamber, followed by incubation with the appropriate fluorochrome-conjugated secondary antibody for 2 h at room temperature. Nuclei were stained with Hoechst 33342 dye . Immunohistochemistry was performed on paraffin-embedded sections subjected to antigen retrieval as described above. After blocking, sections were incubated with primary antibodies at 4 °C overnight in a humid chamber, followed by incubation with biotinylated IgG at RT for 30 min. Detection of the signal was carried out using Vectastain ABC kit  and diaminobenzidine (DAB) substrate kit  according the manufacturer’s instructions. The sections were counterstained with hematoxylin. Microscopy was performed using a fluorescence microscope (Nikon Eclipse E600), Spot RT3 camera and Spot imaging software.Statistical analyses were carried out as previously detailed . Briefly


Objective: There is currently no effective medicine or supplement for clearance of high risk- human papillomavirus (HR-HPV) infections. We have taken a systematic approach evaluating the potential use of AHCC supplementation to support clearance of HR-HPV infections. The primary objective of this research was to evaluate AHCC supplementation to modulation of the host immune system to clear HR-HPV infections from bench to bedside.Methods: Cervical cancer cells, CaSki (HPV16+), HeLa(HPV18+), SiHa(HPV16/18+), and C-33A(HPV−), were treated in vitro with AHCC 0.42 mg/mL daily x7 days then observed x7 days with daily sample collection. A confirmatory study in cervical cancer mouse models, SiHa(HPV16/18+) and C-33A(HPV−), was conducted: mice were divided into three groups per cell line then dosed with AHCC 50 mg/kg/d (N = 10), or vehicle alone (N = 10), or no supplementation (N = 10) for a total of 90 days followed by 30 days of observation. Tumors were measured 3x/week and blood samples collected bi-weekly to evaluate interferon (IFN) alpha(α), beta(β), and gamma(γ) and immunoglobulin G(IgG) by immunoassays. Tumors were evaluated for HR-HPV expression by PCR. Two pilot studies of 10 patients each were conducted in women with confirmed persistent HR-HPV+ infections. The 1st study evaluated AHCC 3g from 5 weeks up to 6 months and 2nd study evaluated AHCC 1g < 8 months. HR-HPV DNA status and the immune panel were monitored at each visit.Results: HR-HPV clearance was observed in vitro and confirmed in the animal studies as a durable response. Four of six (66.7%) patients had confirmed HR-HPV clearance after 3–6 months of AHCC 3g. Similarly, 4 of 9 (44%) patients had confirmed HR-HPV clearance after 7 months of AHCC 1g. Suppression of IFNβ <25 pg/mL was observed in those clearing the HR-HPV infection.Conclusion: Pre-clinical in vitro and in vivo studies demonstrated durable clearance of HR-HPV infections. The preliminary data from the two pilot studies suggested that AHCC supplementation supports the host immune system for successful clearance of HR-HPV infections. A confirmatory phase II randomized, double-blinded, placebo-controlled study is ongoing. Several studies have reported a variety of therapeutic effects, including antioxidant and anticancer activity and improvement of immune response . In animin vitro and in vivo cervical cancer mouse models, and to define the mechanism of clearance of HR-HPV infections with AHCC supplementation. In the initial pilot studies, objectives were to evaluate if AHCC supplementation would modulate the host immune system to clear HR-HPV infections in women with confirmed, persistent HR-HPV infections, to determine duration of AHCC supplementation needed to clear HPV infections and confirm durability of response.This manuscript presents over a decade of research from bench to bedside to test the hypothesis that AHCC supplementation will modulate the host immune system to effectively clear chronic, persistent HR-HPV infections. There is currently no cure for persistent HR-HPV infections. Hence, confirmation of preclinical findings was essential before reporting of this data to demonstrate translation to humans. First preclinical study objectives were to evaluate if AHCC supplementation would clear HR-HPV expression AHCC was generously provided by Amino Up Chemical Co, Ltd. . Fetal bovine serum (FBS) and trypsin-EDTA were purchased from GIBCO Invitrogen Co. . The 3--2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich Co. . Ready–to–use sandwich ELISA kits for the detection of mouse IFN-alpha/beta, IFN-gamma were purchased from eBioscience . ELISA kit for the detection of mouse IgG1 was purchased from GenWay Biotech, Inc. .2 culture flasks in 5% CO2 in air at 37°C to 90% confluence. Cell lines used for this study were maintained for <15 passages to prevent major changes in cell line characteristics.All human cervical cancer cell lines C-33A (HPV negative) and CaSKi (HPV16 positive), HeLa (HPV 18 positive), and SiHa (HPV16 & HPV18 positive) were obtained from the American Type Culture Collection . The SiHa squamous cell carcinoma, HeLa adenocarcinoma, and C-33A carcinoma cell lines were propagated in a media consisting of EMEM with 2 mM L-glutamine and Earl's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate and 10% FBS. The CaSKi epidermoid carcinoma cell line was propagated in a media consisting of RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium carbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 10% FBS. All cell lines were grown in 75-cmAHCC 40 mg/mL stock solution was prepared by dissolving 600 mg of AHCC powder in 15 mL phosphate buffered saline (PBS) and filters sterilized with 0.2 μm syringe filter. All additional dilutions were completed as required with the respective cell culture media for each cell line. The MTT stock solution was prepared by dissolving 54 mg of MTT in 20 mL PBS to achieve a final concentration of 0.3 mg/mL. Fresh standards and dilutions were made for each experiment.20 (inhibitory concentration to achieve 20% cell death), and IC50 for AHCC and each cell line were calculated  and C-33A (HPV negative) were treated in T25 flasks, one time with AHCC (0.42 mg/mL) for 72 h  every 24 h, then observed for an additional 7 days. Untreated cells served as a control. Cells were harvested once every 24 h for total of 14 days during supplementation and observation phases of the study. DNA was extracted from all samples and used for PCR as described below.Growth inhibition assays were conducted as previously described . The IC2lculated . During for 72 h . The IC2N = 60 for two cell lines). There were three arms of this study: AHCC supplementation arm (N = 10), no supplementation control arm (N = 10) and a vehicle (autoclaved water) control arm (N = 10). The no supplementation control arm had no study intervention; Following traditional cancer chemoprevention models, the AHCC supplementation arm received an oral dose  and the vehicle control arm received an oral dose of autoclaved water  per day starting on day zero and continued until completion of the study (day 90) to evaluate if AHCC also has role in prevention of cancer growth. The dose of AHCC was selected based current estimated maximum recommended dosage of 3 g total daily dose as instructed by the manufacturer, assuming 100% bioavailability and using an estimated total blood volume of an average adult of 60 kg. It is also the dose used in previous AHCC studies in combination with chemotherapy. SiHa cells (0.5 × 106) and C33A cells (0.5 × 106) were dispersed in PBS with 20% matrigel and injected subcutaneously in female athymic/immune-suppressed mice on day 8 after 1 week of AHCC supplementation. Each mouse grew one tumor on the dorsal surface. Tumor measurements were obtained three times per week with electronic Vernier calipers . Mice were monitored daily for signs/symptoms of morbidity including but not limited to lethargy, weight loss, anorexia, or hunching. Mice were sacrificed via CO2 inhalation followed by cervical dislocation, when tumor diameter was >15 mm2, and if a 10% or greater decrease in body weight was found during the study period. Each group was subdivided into two groups A & B. Blood sample of 1% of the body weight, from all the mice of all groups were collected from their facial vein every 14 days in an EDTA containing tube, to carry on the assay for expression of different immune markers at the end of the study. The whole blood was centrifuged; the separated serums were collected in a tube and were stored at negative 20°C freezer. The serums of sub group A were tested for IgG1 and IFN γ, and the serums of sub group B were tested for IFNα/β. At the end of the study all the remaining mice were sacrificed, blood samples were collected by cardiac puncture, serums were separated, and stored at negative 20°C. Tumors were collected in cryovials and were stored at negative 80°C for further study in PCR studies to evaluate HR-HPV status as described below.There currently is not an ideal mouse model for evaluating HR-HPV infections alone. A cervical cancer model was selected as best surrogate for evaluating elimination of HR-HPV infections because the SiHa human cervical cancer cell line expresses both HPV16 and HPV18. The C33 tumor model was selected as a HPV negative control for the study. The protocol was reviewed and approved by the institutional animal welfare committee (AWC) prior to initiating any animal work. For this study, 70 female athymic/immune-suppressed mice, 6–8 weeks old, were obtained from Harlan Laboratories . All the mice weighed about 22–26 g; they were maintained five per cage in specific pathogen free (SPF) barrier room, with a temperature of 22 ± 3 and 45 ± 3°C RH%. There was free access to autoclaved food and reverse osmosis autoclaved water. The experiment procedures and the handling of the mice were in strict accordance with the guide for the care and use of laboratory animals. There was a group of healthy controls which had 10 athymic/immune-suppressed mice in it, they were not treated throughout the study nor were they injected with any cancer cell lines and served as controls for immune marker monitoring. The remaining mice were divided into three groups of 10 for each cell line  according to manufacturer's protocol. The polymerase chain reaction (PCR) was used to detect the expression of human papilloma virus (HPV) gene as per the manufacturer's protocol using Promega-Master Mix Kit . The analyses were performed on 500 ng of purified DNA of treated and untreated cell and tumor samples as described below. PCR reaction contained 500 ng of DNA, 4 μL of Master Mix, and 4 μL of each consensus primer . Thirty cycles of start denaturation (95°C for 4 min), denaturation (94°C-1 min), annealing (55°C-1 min), and extension (72°C-1.5 min) were performed on a BioRad thermocycler. β-actin was used as the DNA loading control. After the completion of the reaction, 10 μL each of all PCR products were resolved on 1.5% agarose gel with ethidium bromide against 100 bp DNA ladder. The image was captured under UV illumination using Kodak Imaging Station . The primer sequence of MY11 is DNA 5′ to 3′-GCM CAG GGW CAT AAY AAT GG and MY09 is DNA 5′ to 3′-CGT CCM ARR GGA WAG TGA TC. The sequence of β-actin forward primer is AAC TGG GAC GAC ATC GAG AA and the sequence of β-actin reverse primer is AGA GGC GTA CAG GGA TAG CA.Following the respective time point supplementation regimens of base line, week 2, week 4, week 6 and end time point, blood samples were collected from mice and serum was separated and stored at −20°C for ELISA assays for the detection of IgG, IFNα/β, and IFNγ. For each series of immune markers determinations, a standard curve was constructed with known concentrations of these markers according to the manufacturer's protocol. Sandwich ELISAs for the detection of total IgG, IFN α/β, and IFN γ were performed according to the manufacturer's protocol. Serum concentrations of these three markers were calculated from standard graphs and were compared with base line concentrations. The percent increase and/or decrease in the concentration of IgG, IFNα/β, and IFNγ were calculated.The pilot studies and study amendments (HSC-MS-12-0851) were approved by the Institutional Review Board of the University of Texas Health Science Center at Houston. Two pilot studies were conducted in women over the age of 30 with documented persistent HR-HPV infections for over 2 years or more, had laboratory values all within normal limits, normal histology up to CIN2 as documented by the primary gynecologist's records, and deemed otherwise healthy were enrolled on study. All patients signed informed consent to participate in each of the respective pilot studies. Patient demographic information was collected including: number of lifetime sexual partners, contraception methods, and periodic pregnancy testing throughout the study. Participants received either AHCC 1g by mouth once daily on an empty stomach for 6 months up to 8 months or AHCC 3g by mouth once daily on an empty stomach for 5 weeks up to 6 months to clear HR-HPV. HR-HPV testing was completed with Cervista HPV DNA testing  in the AHCC 3g study. In the AHCC 1g study the patient samples were initially tested with HPV E6/E7 RNA testing  and then all HPV RNA negative results confirmed or COBAS HPV DNA testing . A durable response was defined on remained HR-HPV negative by both HPV DNA and HPV RNA methods for at least 3 months during the observation period after stopping the AHCC supplementation. In both studies immune markers including IgG and IFN α/β/γ were monitored with ELISA assays as described above.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826013/). Briefly, an E-value that is equal to number of animals minus number of groups is calculated and ensured to be between 10 and 20. In this study there was independent comparisons of HR-HPV status as well as immune response between group 1 vs. 2 (AHCC), 1 vs. 3 (vehicle control) and 1 vs. 4 (no supplementation control), thus E = (10 × 2)−2 = 18, which is within range. The student t-test analysis was used to evaluate the differences between the study arms that responded verses those that did not with p-values less than or equal to 0.05 considered significant.The “resource equation” method was employed to determine animal study sample size estimation , for AHCC ranged from 2.3 to 4 mg/mL in the four human cervical cancer cell lines however this is well above clinically achievable systemic concentrations. These results are summarized in In the growth inhibition assays supplementation with AHCC for 72 h did not achieve any cell growth inhibition in any of the four human cervical cancer cell lines tested at clinically relevant concentration of 0.42 mg/mL. However, potential direct cytotoxicity was observed the concentration to achieve 50% growth inhibition (IC+/18+) compared to C33a HPV negative control are summarized in Supplementation with AHCC 0.42 mg/mL one-time dose for 72-h incubation, suppressed HR-HPV expression with first 24 h, but then HR-HPV expression was recovered by 48 h. The results for the C33a (negative control) and SiHa (HPV16 & HPV18 positive) cell lines are summarized in in vivo confirmatory mouse studies, AHCC 50 mg/kg by mouth once daily for 90 days was associated with clearance of the expression of HR-HPV that was sustained after 30 days off supplementation. These results are summarized in p < 0.05). IFN-β immune response is summarized in In the A separate AHCC 1g follow up pilot study was initiated to determine if a lower dose of AHCC supplementation would also be effective. Patients received AHCC 1g daily on an empty stomach for at least 6 and up to 8 months. Ten women with confirmed persistent HR-HPV infections for >2 years were enrolled. Patient demographics are summarized in in vitro in the panel of human cervical cancer cell lines, animal studies were completed that also demonstrated successful, durable elimination of HR-HPV after completing AHCC supplementation. Finally, in two “proof of concept” pilot studies of daily AHCC supplementation successful elimination of HR-HPV was achieved that was durable response. Both the animal and human data suggests the mechanism AHCC supplementation supports the host immune system to clear HPV infections is attributed to the modulation of the expression and signaling of IFNβ that is known to be elevated in chronic viral infections .The presented bench-to-bedside research provides step-wise data to support the hypothesis that AHCC supplementation modulates host immune system, specifically via suppression of elevated IFNβ levels, to effectively clear chronic, persistent HR-HPV infections. After observing elimination of HR-HPV In previous CMV mouse model studies have demonstrated that IFNβ, type I interferon, is associated with virulence of the infection with significantly elevated levels in chronic/persistent viral infections , 17. TheThere are some limitations to data presented. First, in the preclinical setting there is no established HR-HPV animal model which has also been a challenge for in the pre-clinical development of HPV vaccines. As a surrogate a human tumor infected with HR-HPV was employed to determine if the HR-HPV infection could be cleared with AHCC supplementation and was durable response after AHCC supplementation was stopped. In addition, in both the pre-clinical and clinical studies it would be ideal to confirm eradication with quantitative PCR methods as well as evaluate other immune markers such as CD8+ T-cells or natural killer (NK) cells but due to limited funding and being exploratory study, an exhaustive immune panel evaluation could not be completed. However, in the ongoing phase II confirmation study these studies are being evaluated.There are limited effective treatment options for clearing HR-HPV infections; majority being local treatment modalities is to alleviate symptoms and remove symptomatic lesions that often reoccur. Fortunately, most HR-HPV infections are cleared within 6–18 months on their own without interventions; only ~10% of women will suffer from persistent HR-HPV infections , 24. To This bench to bedsides studies are the first preliminary study to demonstrate AHCC supplementation modulates the host immune response to effectively clear of HR-HPV infections. Similar to other chronic viral infections IFNβ suppression was confirmed as marker of durable clearance of HR-HPV infection. AHCC 3g achieved response slightly faster and more consistently than the AHCC 1g. These preliminary findings are very encouraging and await confirmation from the ongoing phase II randomized, double-blinded, placebo controlled study in 50 women with confirmed HR-HPV+ infections. Futures studies will focus on the potential role for AHCC supplementation in combination with chemotherapy for the treatment of HR-HPV-related cancers.The datasets generated for this study are available on request to the corresponding author.JS contributed to conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, writing—original draft and editing. LM and AG contributed to data curation, investigation, formal analysis, methodology, validation, writing—original draft, review and editing. BR and MB contributed to data curation, investigation, writing—review and editing. JF contributed to methodology, investigation, writing—review and editing. JL and TB contributed to investigation, writing—review and editing. YB and RO contributed to methodology, supervision, writing—review and editing.JS has been principal investigator on unrestricted research grants from Amino Up Chemical Company to the institution for preliminary preclinical studies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.