The KP has been reported to be elevated under inflammatory conditions such as cancers and that its activity suppresses immune surveillance. Dysregulation of the KP has previously been reported implicated in BrCa. This review aims to discuss and provide an update on the current mechanisms involved in KP-mediated immune suppression and cancer growth. Furthermore, we also provide a summary on 58 studies about the involvement of the KP and BrCa and five clinical trials targeting KP enzymes and their outcome.Breast cancer (BrCa) is the leading cause of cancer incidence and mortality in women worldwide. While BrCa treatment has been shown to be highly successful if detected at an early stage, there are few effective strategies to treat metastatic tumours. Hence, metastasis remains the main cause in most of BrCa deaths, highlighting the need for new approaches in this group of patients. Immunotherapy has been gaining attention as a new treatment for BrCa metastasis and the kynurenine pathway (KP) has been suggested as one of the potential targets. The KP is the major biochemical pathway in tryptophan (TRP) metabolism, catabolising TRP to nicotinamide adenine dinucleotide (NAD Breast cancer (BrCa) is the leading cause of cancer incidence worldwide with 2.26 million cases in 2020 alone or 11.7% of total cancer cases . TreatmeDespite the combination of advancements in early screening and detection and targeted therapy as highlighted above, metastasis remains a cause of more than 90% of BrCa deaths . ImmunotTRP is one of the essential amino acids that is not endogenously synthesised and needs to be obtained from the diet for protein synthesis . Once inTDO is expressed mainly in the liver  and othe+ product, or into other KP intermediates in a multi-branched arrangement, mediated by enzymes and their inducers acting at the different levels of the pathway [As mentioned above, TRP can be metabolised into the KP producing NAD pathway , 51  by 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD) [The preferred catabolic route for KYN in the brain is to produce KYNA by KATs under normal physiological condition . Due to  (ACMSD) .+, a co-factor required in many biochemical processes. In some mammals, the KP can supply sufficient NAD+ in the absence of any dietary niacin intake [As mentioned above, the KP has a major role in physiological processes through the supply of NADn intake . Anothern intake . It has n intake , 63.Initially, the KP was simply thought to function as an immunomodulatory mechanism by allowing cells to deplete TRP from the intracellular pool or local microenvironment and, therefore, lead to the suppression of T-cell function and growth . AccumulThe involvement of the KP in cancer was first observed in early studies where cancer patients were reported to have higher KP activity , 71. As + T-cell populations through the caspase activation pathway while inhibiting CD8+ T-cell expansion and activation [+ T cells [+ when catabolised. This NAD+ is then utilised in DNA repair, essentially reducing the apoptosis that was triggered by chemotherapeutic drugs [The second immune suppression mechanism of the KP involves its metabolites and their interaction with immune T cells and tumours Fig. . Notablytivation , 87. Addtivation . On the  T cells , 89. Apaic drugs .A last immune-suppressive mechanism of the KP is mediated through the interaction between some KP metabolites and the ligand-activated transcription factor, the AhR. The AhR is expressed in all cell types, and its activation can trigger various biological pathways regulating the immune response and various cellular functions . Other tThe KYNA triggered observed feedback loop is produced by the activation of the Interleukin-4 induced gene 1 (IL4I1) rather than the KP. Sadik et al. showed that melanoma has high expression of IL4I1 producing KYNA, and KYNA activation of the AhR enhanced tumour cell motility and T-cell proliferation . ElevateWe conducted a search on PubMed using the keywords “kynurenine” and “breast cancer” and we found 58 studies that looked at the KP in BrCa. These studies and their outcomes are summarised in Table The possibility that TRP metabolism may have a role in BrCa was alluded to in early studies from the 1970’s by measurement of 3HAA, 3HK, AA, KYN, KYNA and XA in the urine of patients following ingestion of TRP –99. Two The advancements in analytical chemistry and molecular biology techniques may have led to an increased number of studies on the KP in BrCa after the year 2000. In particular, analytical chemistry techniques such as high-performance liquid chromatography (HPLC) have provided high-resolution separation and quantitation of components from a biological matrix with greater accuracy, sensitivity and reproducibility of results, which has made it an appropriate method to quantify chemical constituents in biological samples . HPLC haSince the early 2000’s, there were 40 studies examining the role of IDO1 in BrCa that includes the impact of chemotherapy on IDO1 activity and inhibition of IDO1 itself. As noted above, IDO1 is a significant rate-limiting enzyme that regulates the production of KP metabolites and is generally highly expressed in multiple human cancers . AlthougInterestingly, chemotherapy treatment reduced the activity of IDO1 in serum samples of BrCa patients and animal model study , 118–122The above studies support the notion that IDO1 is elevated in tumours to suppress immune surveillance and favour tumour growth, suggesting IDO1 as a potential therapeutic target to treat BrCa. It is also important to note that there were studies showing either an increase in IDO1 activity –133 or nIDO1 has emerged as an attractive pharmacological target as the enzyme has been well characterised and has high affinity for TRP among the small number of TRP catabolising enzymes . Given that it is the rate-limiting enzyme, inhibition of its activity may potentially block or reduce the activity of the whole KP, and its inhibitory action can be measured indirectly in the blood. As most of human cancers, including BrCa, overexpress IDO1, the validation of IDO1 inhibitor efficacy in enhancing anti-tumour immune activity could be translated for clinical use. With the positive outcome from the demonstrated efficacy of limiting tumour growth with combination treatment of IDO1 inhibition and chemotherapy drug(s) in in vivo and in vivo studies, 97 different clinical trials have been reported in ClinicalTrials.gov examining combination therapy in patients with cancers . Among tEpacadostat has been shown to have the greatest efficacy in inhibiting IDO1 activity , 146 andIndoximod is the most studied IDO1 inhibitor in in vivo studies. It mimics TRP in reversing IDO1-mediated inhibition of the mammalian target of rapamycin complex 1 (mTORC1) , 151. mTn = 30) were found to have longer progression free survival and overall survival than patients with low IDO1 expressing tumours (n = 22), the differences were not statistically significant. A later phase I/II trial, NCT01042535, examined the efficacy of combining indoximod with an adenovirus-p53 transduced dendritic cell vaccine in the treatment of patients with metastatic BrCa. Of the 39 patients enroled, 22 patients who obtained more than two cycles of post vaccination therapy, which included chemotherapy drugs, were evaluated for response. One patient achieved complete response, seven partial response and one remained with stable disease. Among these nine patients that continued into Phase II, five patients showed a 10% increase in activated CD8 + cells in their blood count. Despite a favourable outcome, this combination therapy did not achieve the pre-set threshold of a 20% objective response rate [Although patients with high IDO1 expressing tumours  or placebo plus pembrolizumab (n = 352) (4.7 months and 4.9 months respectively). In addition to this, treatment-related adverse events were reported in 10% of patients receiving the combination treatment and 9% of patients receiving the placebo [In this review, we have presented strong evidence that the KP is dysregulated in BrCa and that several of its enzymes may be potential targets to enhance the anti-tumoral immune response. In particular, the positive outcomes from animal studies have led to multiple clinical trials examining the potential of combining IDO1 inhibitor in combination treatments for cancer patients, including six clinical trials with BrCa patients. However, these combined therapies did not improve patient outcomes as compared to monotherapy. These discouraging results echoed the failed phase I/II clinical trial evaluating epacadostat plus pembrolizumab in malignant melanoma. There were no significant differences between the treatment groups for progression free survival in epacadostat and pembrolizumab arm ( placebo . It is n placebo . In the  placebo .There are also some concerns about IDO1 inhibition that would need to be addressed in future studies. The first research question to address is whether a compensatory mechanism is activated when IDO1 is inhibited. Considering that TDO and IDO2 are in the same node of the pathway, inhibition of IDO1 may lead to a feedback mechanism to overexpress TDO and/or IDO2. TDO may likely be the prime substitute enzyme as it is not only expressed constitutively , 163 butOverall, the KP has been shown to have a strong association with BrCa and may play a role in the pathology of this cancer. An in-depth understanding of the pathway could either lead to better development of KP inhibitors or a monitoring tool for treatment response for BrCa. As demonstrated in a recent publication of a clinical trial examining the combination of Indoximod with checkpoint inhibitors, the combination therapy was well tolerated and showed anti-tumour efficacy in patients with melanoma . Ongoing


In the left TA, the activity decreased in the sitting posture during non-GI (p = 0.004) and GI-1 (p = 0.009) in comparison to GI-2. No differences were found for GM. The subjective level of imagination difficulty improved for GI-2 in comparison to GI-1 in both postures (p < 0.001). Previous sensorimotor experience with real gait execution and sitting posture potentiate TA activity decrease during GI. These findings contribute to the understanding of neural mechanisms beyond GI. This study explores how gait imagery (GI) influences lower-limb muscle activity with respect to posture and previous walking experience. We utilized surface electromyography (sEMG) in 36 healthy young individuals aged 24 (±1.1) years to identify muscle activity during a non-gait imagery task (non-GI), as well as GI tasks before (GI-1) and after the execution of walking (GI-2), with assessments performed in both sitting and standing postures. The sEMG was recorded on both lower limbs on the tibialis anterior (TA) and on the gastrocnemius medialis (GM) for all tested tasks. As a result, a significant muscle activity decrease was found in the right TA for GI-1 compared to GI-2 in both sitting ( Motor imagery is defined as a purely cognitive process where the individual imagines making a movement and the imagery is not accompanied by any visible manifestation of such a movement. Motor imagery is an active process by which the representation of a certain action is internally reproduced via motor memory, while the movement itself is inhibited and the somatosensory inputs generated during motion are absent ,2,3,4. WMotor imagery manifests similar patterns of neural activity to actual movements ,12,13,14Motor imagery shares the activity of specific neural substrates similarly to during movement execution ,18,19,20Even though the brain activity that is connected to motion imagination has received quite a lot of attention, studies focusing on the changes in the excitability on the spinal level ,27,28 orFrom the motor control perspective, the influence of motor imagery on muscle activity may represent the missing piece of evidence behind the more complex effect of motor imagery as the motoneuron pools of muscles involved in imagined movements receive summations of inputs from both descending neural pathways and from sensory afferents, similarly to motion execution.The fact that movement imagery influences muscle activity has been unambiguously proven by studies pointing to the increased muscle power resulting only from mental training  and the Human gait is considered a complex motor task that involves coordinated muscle activation and balance control, as well as the adaptation of movements to the external environment. The rhythmic complex patterns of the muscle activity required for locomotion are, to a great extent, driven by the neural networks at the spinal level, referred to as central pattern generators (CPGs) ,23,24,25Based on the published studies ,30, it sThe question arising from this topic is whether sitting  or standing  influences the final electromyographic outcome during GI as both these positions provide different somatosensory feedback. The aim of the study was to assess how default sitting or standing posture and immediate previous sensorimotor experience with real walking modulate activity of distal lower-limb muscles during gait imagery in young, healthy individuals.The study involved 36 young healthy individuals (23 women and 13 men). The average age of the participants was 24 (±1.1) years, with a height of 164 (±8.9) cm and a weight of 68 (±12.3) kg. All participants who volunteered to participate in this study were recruited from university students of a master’s program in Physiotherapy. All participants partook in leisure sport activities and none of them were sport professionals. All participants provided informed consent. The research was approved by the local ethics committee . All procedures were performed according to the ethical standards of the Declaration of Helsinki.The criterion for participation in the study was good motor imagery—classified as being at least mark 3 on average, in accordance with the standardized Movement Imagery Questionnaire—Revised (MIQ-R). Therefore, only participants with at least moderate imagery ability were recruited. MIQ-R, which represents a valid and reliable instrument for measuring motor imagery ability in healthy persons ,49, usesAnother criterion for participation in the study was the absence of any acute postinjury condition, a neurological condition, an orthopedic condition, pain, or a cognitive deficit that could in any way restrict or preclude the measurement.The influence of GI on muscle activity was assessed through telemetric sEMG using hybrid bipolar electrodes with default inter-electrode distance  in sitting and standing postures.Muscle activity was recorded from the tibialis anterior (TA) and gastrocnemius medialis (GM) in both lower limbs. The electrodes were placed onto the belly of the respective muscles with self-adhesive straps; then, the palpation of muscle bellies was conducted with sub-maximal isometric contractions according to the standards set by SENIAM . Prior tThe initial postures for all tested situations and participants were always sitting or standing with eyes open, facing the white screen see . The pos1. In an initial posture without imagining any movement (non-GI). To prevent the generation of any other imagery that would be undesirable during this task, the participants imagined singing the song “Happy Birthday”. The participants were asked by the researcher to sing the song in their mind and, after 30 s, were given a signal to stop.2. In an identical position, the participants were instructed to imagine walking along a corridor ) as precisely as possible. Prior to the imagery, all participants saw the corridor, which was outside a laboratory. The instructions given to every participant before the measurement began were as follows: “Imagine yourself walking the corridor you have just seen at your comfortable gait speed”. After 30 s, the participants were given a signal to stop.The participants were subsequently asked to walk at their own comfortable speed along the corridor outside a laboratory.3. The participants took the same initial position as in situations 1 and 2. They were instructed to imagine walking the corridor they had just walked ). The instructions were as follows: “Imagine yourself walking the corridor you have just walked at your comfortable speed”. After 30 s, the participants were given a signal to stop.The time duration of 30 s of imagination reflected the self-reported optimal time duration of stereotype gait imagination.Electromyographic activity was recorded throughout all 3 experimental situations, which are illustratively demonstrated in For situations 2 and 3 , every participant was asked to write the subjectively perceived level of simplicity/difficulty of GI on a four-point scale, where classification 1 referred to significant difficulty, 2 to moderate difficulty, 3 to moderate simplicity and 4 to significant simplicity in imagining the gait.All testing procedures were realized in a quiet room in one day.® Analysis software , where the 10 s in the middle of the record, without artefacts, was evaluated. Data were processed by removing the mean value and half waves were rectified using the root-mean-square algorithm (with a window size of 0.25 and a window overlap of 0.05 s) and were high-pass filtered at 20 Hz. Data were subsequently used to calculate the mean values of muscle activity (EMGrms) for all tested muscles and for all three tested conditions in both the sitting and standing postures. These mean values were statistically analyzed.The processing of electromyographic data was performed in EMGworksp = 0.017.The statistical processing of data was performed using STATISTICA software (version 13.4.0). The differences in muscle activity for the individually tested situations were assessed using Friedman’s ANOVA test. Regarding the non-normal data distribution, the method selected for within-group comparison and the post hoc test was the Wilcoxon test for two paired samples, with Bonferroni’s correction for multiple testing. Considering the number of examined situations, the level of significance after Bonferroni’s correction was determined at The assessment of a subjectively perceived level of difficulty was performed using the Wilcoxon pair test. The assessment concerned a comparison of the subjectively perceived level of simplicity/difficulty of GI before and after the actual gait in both sitting and standing postures.p = 0.004) and compared with the GI-1 (p = 0.009) in the sitting position. The right TA had significantly lower muscle activity in the GI-2 condition in comparison to the GI-1 in both the sitting (p = 0.008) and standing (p = 0.01) positions. No significant differences were found for the GM. The EMGrms values for the individual muscles and situations that were tested are listed in The study results demonstrated significant decreases in muscle activity for the left TA in GI-2 compared with the non-GI condition (p < 0.001) and standing (p < 0.001) postures.The subjective perception of the simplicity of GI-1 was rated lower by participants in both sitting (2.53 ± 0.88) and standing positions (2.88 ± 0.87) in comparison to GI-2 in sitting (3.16 ± 0.72) and standing (3.41 ± 0.76) positions. In our experiment, the subjective perception of GI simplicity significantly improved immediately after the real walking in both sitting  in order to explore mechanisms that might contribute to the overall neural activity behind imagining walking. The GI immediately following the non-GI condition, in both sitting and standing default postures, did not lead to any significant changes in muscle activity in this study. The conclusion that GI does not lead to changes in lower-limb muscle activity was also previously reached in study concerning brain activity during GI tasks .Significant changes in muscle activity were found when the non-imagery condition was compared to imagination after gait execution for both the left and right TA in the sitting position and for right TA in the standing position. In both positions, we found a decrease in sEMG activity. After gait execution, participants even reported an improved subjective perception of imagined gait. According to our results, it seems that only the immediately preceding perceptual sensorimotor experience  resulted in a change in muscle activity during GI and that this experience even improved the subjective inner perception of GI.Studies focused particularly on central nervous system activity point out the facilitatory effect of motor imagery on excitation at the cortical and subcortical level ,12,13,14The sEMG signal reflects the muscle activity, which is determined by the summation of the final motor unit action potentials and their time-course ,52. The The proper mechanisms of muscle activity facilitation or inhibition during motor imagery are not known at present. Processes that inhibit motor action might originate at the cortical, subcortical or spinal level ,18,29,54It appears that the effect of movement imagery on muscle activity is more pronounced within complex movements when the imagined motion is realized from the first-person perspective, and with proprioceptive feedback corresponding to imagined movement ,27,28,29It is possible that gait imagery influences the sEMG activity of target muscles less in comparison to imagery of upper limb motor tasks ,24 and aMcCrea  previousIt is possible that stereotypic gait imagery tasks without any challenge, which were tested in our experiment, do not activate supraspinal motor regions and, subsequently, descendent spinal pathways that could provide a facilitatory neural drive when imagining gait that is sufficient to evoke muscle activity. Furthermore, it seems that the sitting position, in which walking is impossible, might instead have an inhibitory effect on muscle activity during GI, as previously proven .It was previously suggested that a posture congruent with the imagined task facilitates corticomotor, corticospinal and muscle–spinal reflex loop excitability within imagery tasks ,12,18. TA standing posture, which is congruent with real walking, provides more appropriate somatosensory feedback  compared to postures incongruent with gait execution, such as sitting or lying. Based on the previous findings concerning the effect of sensory information perception from the periphery during imagination ,31,41,42When imagining gait in a position in which walking is impossible, the inhibitory effects might dominate over possible facilitatory effects on muscle activity . It is pThe reason that muscle activity changes in the TA are more manifested in the sitting position may be also because it is more of a resting position, where there is no need to simultaneously generate the necessary muscle activity to maintain an upright standing posture. Authors focusing on the application of motor imagery in sports even mention the sitting posture as a more relaxing position, in which it is easier to concentrate on the imagery .The decrease in sEMG amplitude as a consequence of gait imagination was already proven for GI, GI in combination with observation and GI after gait execution . As was The fact is that in our study, muscle activity decreases were present just after gait execution in the default sitting posture. Muscle activity decreases were previously described for GI after previous sensorimotor experience with real gait  and evenSignificant changes, understood here as muscle activity decreases, were only observed in the TA, rather than in the GM. Both muscles contribute significantly to gait execution—the TA contributes to dorsiflexion and the slight supination of the foot, especially the swing phase of the gait cycle  and the The results of our study are limited to young, healthy adults with very good motor imagery skills. A limit may be even considered due to the fact that just distal leg muscles were evaluated. Further research is needed to observe the effects of motor imagery in different populations of patients with movement disorders. Another limitation of this study, like other studies focused on this area, is the fact that it is generally very difficult to quantify the quality of imagery or hidden cognitive strategies and undesirable imagery.Our findings might contribute to the understanding of neural mechanisms of GI and may also provide further insight into the mechanisms beyond motor recovery as a consequence of training in imagination. From the perspective of practical utilization of gait imagery in rehabilitation settings, this study supports the necessity of previous sensorimotor experience with imagined movement and highlights the importance of the default posture during imagination as both these factors have potential to modulate muscle activity.The results of our study show that GI influenced the lower-limb sEMG activity in cases when GI immediately followed previous sensorimotor experience of real gait. GI performed immediately after real walking led to a decrease in TA activity, with this occurring bilaterally in the sitting position and unilaterally in the standing posture. The results prove that even previous experience with imagined movement facilitates the subjective perception of GI. These findings have potential to contribute to the understanding of neural mechanisms beyond GI.


HIV-1 reservoir cells that circulate in peripheral blood during suppressive antiretroviral therapy (ART) have been well characterized, but little is known about the dissemination of HIV-1-infected cells across multiple anatomical tissues, especially the CNS. Here, we performed single-genome, near full-length HIV-1 next-generation sequencing to evaluate the proviral landscape in distinct anatomical compartments, including multiple CNS tissues, from 3 ART-treated participants at autopsy. While lymph nodes and, to a lesser extent, gastrointestinal and genitourinary tissues represented tissue hotspots for the persistence of intact proviruses, we also observed intact proviruses in CNS tissue sections, particularly in the basal ganglia. Multi-compartment dissemination of clonal intact and defective proviral sequences occurred across multiple anatomical tissues, including the CNS, and evidence for the clonal proliferation of HIV-1-infected cells was found in the basal ganglia, in the frontal lobe, in the thalamus and in periventricular white matter. Deep analysis of HIV-1 reservoirs in distinct tissues will be informative for advancing HIV-1 cure strategies. Approximately 39 million people in the world live with HIV infection. Currently available treatments can reduce the amount of virus to near undetectable levels. But they do not eliminate the virus. A reservoir of HIV-infected cells persists during treatment. If treatment stops, these cells can cause rebounding virus levels and a return of symptoms. As a result, patients living with HIV must remain on treatment their entire lives.HIV reservoir cells often do not express viral proteins, making them hard for the immune system to find and destroy. Many of these reservoir cells occur in lymph nodes, which makes them difficult for researchers to access for study. Learning more about where these cells hide in the body may enable scientists to develop new treatments to help eliminate them.Sun et al. show that HIV reservoir cells exist in many body tissues, including the brain. In the experiments, Sun et al. used single HIV genome sequencing to identify HIV genetic sequences in the brain and other body tissues from three recently deceased individuals with HIV. The individuals agreed to donate their tissues for postmortem studies before their deaths. All received antiretroviral therapy until death. The experiments identified functional HIV genetic sequences in lymph nodes and gastrointestinal tissues, known hotspots for HIV-infected cells. Sun et al. also found genetically intact HIV in brain tissue from two of the individuals. The HIV genetic sequences were identical to sequences found in other body tissues. This discovery suggests HIV-infected cells had divided into more HIV-infected cells and spread.The results suggest that cells harboring intact HIV invade the brain and persist there for extended periods during antiretroviral therapy. To eradicate the virus, interventions targeting HIV reservoir cells must be able to reach the brain. This new information may help researchers developing HIV-reservoir targeting drugs decide which candidates will likely be the most effective. Future studies may also shed light on how HIV reaches the brain and how the infected cells escape destruction by immune cells, which may suggest more treatment strategies. Combination antiretroviral therapy has extended the life expectancy of people living with HIV (PLWH) to near-normal levels, but it is clear that antiretroviral therapy (ART), in its present form, does not lead to a sustained, drug-free remission of HIV-1 infection; instead, a long-lived reservoir of virally-infected cells persists despite ART  and can HIV-1 reservoir cells that circulate in the peripheral blood have been well-described in recent years . These cMuch less is currently known about HIV-1 reservoir cells in other body compartments that are more difficult to access for analytic purposes. Lymph nodes and lymphoid tissues in the gastrointestinal tract harbor the vast majority of all lymphocytes and are likely to represent a prime location for the persistence of virally infected cells ; howeverTo investigate the proviral landscape across multiple anatomical tissues, including the CNS, we focused on three participants from whom post-mortem tissue samples were available for HIV-1 research. Tissue samples from the CNS and other organs were collected by a rapid (<24 hr) autopsy after death. The clinical and demographic characteristics of these study participants are shown in Near full-length single-template next-generation HIV-1 proviral sequencing was performed to profile the proviral reservoir landscape at single-molecule resolution in tissue samples. The number of cells analyzed from each organ in each participant is listed in To evaluate HIV-1 persistence in selected tissue compartments, the frequencies of total, intact, and defective HIV-1 proviruses in each tissue were analyzed . Intact Defective proviral sequences were detected in all analyzed tissue samples except for the thalamus from participant 1 . In partA series of prior studies demonstrated large sequence-identical clones of intact and defective proviruses in the peripheral blood of ART-treated study participants. In our subsequent analysis, we studied the dissemination of clonal proviral sequences across different tissues. In participant 1, a total of 24 intact proviral sequences were detected. Two large clones of intact proviruses were observed, one of which included sequences detected in kidney, lymph node, and spleen samples . The othClonal proviral sequences in CNS tissues were detected in all three study participants. In study participant 1, members of a clone of defective proviruses were observed in the basal ganglia and the frontal lobe . In partViral tropism was evaluated based on the env V3 region of the proviruses using the Geno2pheno algorithm. Notably, all proviral sequences containing the env V3 region of participant 1  and 3  were predicted to be CCR5-tropic . In partThe lifelong persistence of viral reservoir cells makes HIV-1 infection an incurable disease that necessitates indefinite antiretroviral suppression therapy. However, the location of HIV-1 viral reservoir cells across different tissues has been difficult to assess in the past, due to the limited availability of tissue samples. Recent studies, pioneered by investigators of the ‘Last Gift Cohort’, have catalyzed investigations to characterize HIV-1 sequences in diverse organ systems, specifically in the CNS . In our HIV-1 can invade the CNS within days after infection, as demonstrated in animal  and in hOur study did not allow to determine which cell types were infected by HIV-1 and responsible for clonal expansion of viral reservoir cells in the CNS; however, it is possible that infected microglia are involved. Microglial cells originate from erythromyeloid progenitors in the yolk sac and colonize the developing CNS during embryogenesis ; these cIn our study, CXCR4-tropic proviruses were exclusively detected in participant 2. Compared to the other two participants who began ART shortly after HIV-1 diagnosis, participant 2 was diagnosed with HIV-1 25 years prior to starting antiretroviral therapy and died 10 months after ART commencement; therefore, viral CXCR4 tropism most likely resulted from ‘coreceptor switch’ frequently occurring during advanced stages of immune deficiency . WhetherOur study has several limitations. Importantly, this work includes only 3 participants, and brain tissues were the only samples available from participant 3. Moreover, due to limited tissue sizes available for investigation, very few cells were assayed from the terminal ileum and testes; prior studies suggested high HIV-1 DNA levels in these 2 tissues during suppressive antiretroviral therapy . AnotherIn sum, this study provides a deep analysis of tissue reservoirs for HIV-1 that includes a detailed assessment of HIV-1 sequences in CNS tissues. Our work supports the persistence of genome-intact HIV-1 in many tissues, including CNS tissues, emphasizing the difficulties in finding strategies to effectively eliminate HIV-1 from the human body in clinical settings.HIV-1-infected study participants were recruited at the Massachusetts General Hospital (MGH) and the Brigham and Women’s Hospital in Boston, MA. Fresh tissues were sampled during routine autopsy according to protocols approved by the Institutional Review Board and cryopreserved for future study according to standard protocols. The clinical characteristics of study participants are summarized in Tissue samples were dissected and subjected to genomic DNA extraction using the DNeasy Blood and Tissue Kit . HIV-1 DNA was analyzed by the Intact Proviral DNA Assay (IPDA), using primers and probes described previously . PCR washttps://github.com/BWH-Lichterfeld-Lab/Intactness-Pipeline; Genomic DNA diluted to single HIV-1 genome levels based on Poisson distribution statistics and IPDA results was subjected to near full-genome HIV-1 amplification using a one-amplicon approach . PCR prohttps://coreceptor.geno2pheno.org/). HIV-1 tropism was classified as ‘CCR5’ if the false-positive rate (FPR) predicted by Geno2pheno was >2%, however, 92% of our proviral sequences meeting this definition had a FPR score >10%; proviruses were considered ‘CXCR4-tropic’ if FPR was <2%.Data are summarized as bar graphs. Phylogenetic relationships were evaluated using maximum-likelihood phylogenetic trees. Images were prepared using Adobe Illustrator. HIV-1 tropism was computationally inferred using Geno2pheno , HIV-1 persists in cellular reservoirs in vivo. The viral reservoir in circulating memory T cells has been well characterized, in part due to the ability to safely obtain blood via peripheral phlebotomy from people living with HIV-1 infection (PWH). Tissue reservoirs in PWH are more difficult to sample and are less well understood. In this small (n=3) autopsy study, Sun and colleagues use an advanced genetic sequencing technique to characterize HIV-1 that persists in human tissues despite antiretroviral therapy. The authors describe isolation and genetic characterization of HIV-1 reservoirs from a variety of tissues including the central nervous system (CNS) obtained from three recently deceased individuals at autopsy. They identified clonally expanded proviruses in the CNS in all three individuals.Strengths of the work include the study of human tissues that are under-studied and difficult to access, and the sophisticated near-full length sequencing technique that allows for inferences about genetic intactness and clonality of proviruses. The small sample size (n=3) is a drawback. Furthermore, two individuals were on ART for just one year at the time of autopsy and had T cells compatible with AIDS, and one of these individuals had a low-level detectable viral load . This makes generalizability of these results to PWH who have been on ART for years or decades and have achieved durable viral suppression and immune reconstitution difficult.While anatomic tissue compartment and CNS region accompany these PCR results, it is unclear which cell types these viruses persist in. As the authors point out, it is possible that these reservoir cells might have been infiltrating T cells from blood present at the time of autopsy tissue sampling. Cell type identification would greatly enhance the impact of this work. Overall, this small, thoughtful study contributes to our understanding of the tissue distribution of persistent HIV-1, and informs the ongoing search for viral eradication. The authors were trying to survey reservoir viral sequences in different anatomical sites in the body, with the brain being of special interest. This is a study that is technically demanding and here is well done, providing insights that prompt new and more sophisticated questions.The authors use end-point dilution PCR to identify individual proviruses that can then be sequenced with high accuracy. These are high quality data sets but given the technical requirements of this approach the number of sequenced proviruses is limiting given the scope of questions this study addresses. Nonetheless, there is a lot of data here to draw many useful conclusions.It will be important to realize how clones of infected T cells can move around the body, including into the CNS compartment. It will also be important to remember that there are limits in sampling depth of proviruses in any one tissue meaning the failure to detect something has a limit in sensitivity of detection when trying to interpret a negative result.As noted in the next section, it is important to emphasize that there is another entry phenotype beyond X4 that will ultimately be important in interpreting these results. Macrophage-tropic viruses are often found in the CNS compartment and it will be important to understand whether these CNS-derived sequences are macrophage-tropic viruses there infecting macrophages and microglia or if they are all T-tropic viruses brought in by wandering infected T cells (or both). The following is the authors’ response to the original reviews.We are very grateful to the reviewers for their insightful and detailed analysis of our work, in particular to reviewer 2. We also would like to thank the Elife editorial team for organizing this form of public review and debate, which we believe will be of interest to the science community.Reviewer #1 (Public Review):Despite durable viral suppression by antiretroviral therapy (ART), HIV-1 persists in cellular reservoirs in vivo. The viral reservoir in circulating memory T cells has been well characterized, in part due to the ability to safely obtain blood via peripheral phlebotomy from people living with HIV-1 infection (PWH). Tissue reservoirs in PWH are more difficult to sample and are less well understood. Sun and colleagues describe isolation and genetic characterization of HIV-1 reservoirs from a variety of tissues including the central nervous system (CNS) obtained from three recently deceased individuals at autopsy. They identified clonally expanded proviruses in the CNS in all three individuals.Strengths of the work include the study of human tissues that are under-studied and difficult to access, and the sophisticated near-full length sequencing technique that allows for inferences about genetic intactness and clonality of proviruses. The small sample size (n=3) is a drawback. Furthermore, two individuals were on ART for just one year at the time of autopsy and had T cells compatible with AIDS, and one of these individuals had a low-level detectable viral load . This makes generalizability of these results to PWH who have been on ART for years or decades and have achieved durable viral suppression and immune reconstitution difficult.https://pubmed.ncbi.nlm.nih.gov/35867351/https://pubmed.ncbi.nlm.nih.gov/37184401/While anatomic tissue compartment and CNS region accompany these PCR results, it is unclear which cell types these viruses persist in. As the authors point out, it is possible that these reservoir cells might have been infiltrating T cells from blood present at the time of autopsy tissue sampling. Cell type identification would greatly enhance the impact of this work. Several other groups have undergone similar studies (with similar results) using autopsy samples (links below). These studies included more individuals, but did not make use of the near-full length sequencing described here. In particular, the Last Gift cohort, based at UCSD and led by Sara Gianella and Davey Smith, has established protocols for tissue sampling duringautopsy performed soon after death. We agree with reviewer 1 that studies to identify specific cell types that harbor intact HIV-1 in individual tissue compartments would be very informative; our group has recently initiated such studies.Overall, this small, thoughtful study contributes to our understanding of the tissue distribution of persistent HIV-1, and informs the ongoing search for viral eradication.We thank reviewer 1 for these encouraging remarks.Reviewer #2 (Public Review):The manuscript by Sun et al. applies the powerful technology of profiling viral DNA sequences in numerous anatomical sites in autopsy samples from participants who maintained their antiviral therapy up to the time of death. The sequencing is of high quality in using end-point dilution PCR to generate individual viral genomes. There is a thoughtful discussion, although there are points that we disagree with. This is an important data set that increases the scope of how the field thinks about the latent reservoir with a new look at the potential of a reservoir within the CNS.We greatly appreciate the comments by reviewer 2 and would like to thank them for their detailed and very knowledgeable analysis of this paper.1. The participants are very different in their exposure to HIV replication and disease progression. Participant 1 appears to have been on ART for most of the time after diagnosis of infection (16 years) and died with a high CD4 T cell count. The other two participants had only one year on ART and died with relatively low CD4 T cell counts (under 200). This could lead to differences in the nature of the reservoir. In this regard, the amount of DNA per million cells appears to be about 10-fold lower across the compartments sampled for participant 1. Also, one might expect fewer intact proviruses surviving after 16 years on ART compared to only 1 year on ART. The depth of sampling may be too limited and the number of participants too few to assess if these differences are features of these participants because of their different exposures to HIV replication. On the positive side, finding similarities across these big differences in participant profiles does reinforce the generalizability of the observations.Many thanks for pointing this out. We also noticed that the total number of HIV-1 proviruses is smaller in our study participant 1 (who had been on ART for 16 years), compared to study persons 2 and 3 with more limited treatment durations (1-2 years), however, due to the small number of study persons, we think we cannot use these results for inferring how treatment duration influences viral reservoir size in tissues.1. The following analysis will be limited by sampling depth but where possible it would be interesting to compare the ratio of intact to defective DNA. A sanctuary might allow greater persistence of cells with intact viral DNA even without viral replication (i.e. reduced immune surveillance). Detecting one or two intact proviruses in a tissue sample does not lend itself to a level of precision to address this question, but statistical tests could be applied to infer when there is sampling of 5 or more intact proviruses to determine if their frequency as a ratio of total DNA in different anatomical sites is similar or different. This would allow adjustment for the different amount of viral DNA in different compartments while addressing the question of the frequency of intact versus defective proviruses. One complication in this analysis is if there was clonal expansion of a cell with an intact genome which would represent a fortuitous overrepresentation intact genomes in that compartment.We have performed the analysis suggested by reviewer 2 and included a diagram reflecting the ratio of intact/defective proviruses as a new supplemental figure . Unfortunately, we do not feel comfortable to draw any real conclusions from this additional analysis; the sample sizes are simply too limited.1. The key point of this work is that the participants were on therapy up to the time of death . The predominance of defective genomes is consistent with this assumption. Is there data from untreated infections to compare to as a signature of whether the viral DNA population was under selective pressure from therapy or not? Presumably untreated infections contain more intact DNA relative to total DNA. This would represent independent evidence that therapy was in place.We agree that an analysis of autopsy samples from untreated persons living with HIV-1 would be of great interest, and are actively collaborating with neuropathologists from multiple sites to obtain such samples. Yet, we are not convinced that selection pressure on reservoir cells during ART can be appropriately identified through quantitative virological assays. Rather, we feel that the selection of proviruses can be best assessed when qualitative parameters, including proviral integration sites and their position relative to host epigenetic chromatin features, are evaluated.1. There are several points in Figure 5 to raise about V3 loop sequences. The analysis includes a large number of "undetermined" sequences that did not have a V3 loop sequence to evaluate. We would argue it is a fair assumption that the deleted proviruses have the same distribution of X4 and R5 sequences as the ones that have a V3 sequence to evaluate. In this view it would be possible to exclude the sequences for which there is no data and just look at the ratio of X4 and R5 in the different compartments, specifically does this ratio change in a statistically significant way in different compartments? The authors use "CCR5 and non-CCR5" as the two entry phenotypes. The evidence is pretty strong that the "other" coreceptor the virus routinely uses is CXCR4, and G2P is providing the FPR for X4 viruses. Perhaps the authors are trying to create some space for other coreceptors on microglia, but we are pretty sure what they are measuring is X4 viruses, especially in this late disease state of participant 2. Finally, we have previously observed that the G2P FPR score of <2 is a strong indicator of being X4, FPR scores between 2 and 10 have a 50% chance of being X4, and FPR scores above 10 are reliably R5 (PMID27226378). In addition, we observed that X4 viruses form distinct phylogenetic lineages. The authors might consider these features of X4 viruses in the evaluation of their sequences. Specifically, it would be helpful to incorporate the FPR scores of the reported X4 viruses.Many thanks for these thoughts. We have now included FPR scores for all sequences and considered sequences with FPR score <2 as X4-tropic. Among 497 proviral sequences derived from all three participants, only 14 proviral sequences had FPR scores between 2 and 10 and their tropism was classified as CCR5 in the new Figure 5. We agree that viral tropism analysis of proviral sequences from the CNS would be of particular interest for study subject 2; however, most brain-derived sequences from that person had large deletions in the env region, precluding an analysis of viral tropism.1. We have puzzled over the many reports of different cell types in the CNS being infected. When we examined these cell types (both as primary cells and as iPSC-derived cells), all cells could be infected with a version of HIV that had the promiscuous VSV-G protein on the virus surface as a pseudotype. However, only macrophages and microglia could be infected using the HIV Env protein, and then only if it was the M-tropic version and not the T-tropic version (PMID35975998). RNAseq analysis was consistent with this biological readout in that only macrophages and microglia expressed CD4, neurons and astrocytes do not. From the virology point of view, astrocytes are no more infectable than neurons.We appreciate these comments. As described in our discussion, we agree that the role of astrocytes as target cells for HIV-1 infection is highly controversial; we look forward to future opportunities to evaluate HIV sequences in sorted astrocytes from autopsy tissues.1. The brain gets exposed to virus from the earliest stages of infection but this is not synonymous with viral replication. Most of the time there is virus in the CSF but it is present at 1-10% of the level of viral load in the blood and phylogenetically it looks like the virus in the blood, most consistent with trafficking T cells, some of which are infected (PMID25811757). The fact that the virus in the blood is almost always T cell-tropic in needing a high density of CD4 for entry makes it unlikely that monocytes are infected (with their low density of CD4) and thus are not the source of virus found in the CNS. It seems much more likely that infected T cells are the "Trojan Horse" carrying virus into the CNS.We appreciate the reviewer’s referral to Greek mythology and agree that the hypothesis of infected T cells acting as “Trojan horses” is more intuitive and better supported by available data. We have adjusted our discussion accordingly.1. While all participants were taking antiretroviral therapy at the time of their death, they were not all suppressed when the tissues were collected. The authors are careful not to mention "suppressive ART" in the text, which is appreciated. However, the title should be changed to also reflect this fact.Thanks for pointing this out. From our perspective, ART is never fully suppressive, as low-level viremia  is detectable in almost all ART-treated persons. As such, it is not clear to us that “suppressive” necessarily implies suppression below the detection limits of commercial PCRs. We argue that ART can also be suppressive when plasma viral loads are in the range of 100 copies/ml, as they are in our study subject 3. Nevertheless, we have changed the title to avoid confusion.Reviewer #1 (Recommendations For The Authors):I encourage the authors to compare their autopsy and tissue sampling procedures to those used by The Last Gift researchers and consider including references to this ongoing study. If the authors plan to continue in this line of research, the field would greatly benefit from a collaboration that would bring together their excellent and advanced PCR technique with the larger sample size offered by The Last Gift. Lastly, is there some way to simultaneously determine cell type when NFL sequencing is performed?We look forward to collaborating with investigators from the Last Gift Cohort in the future and have integrated additional references in the manuscript to acknowledge their work. At the current stage of technology development, we think that sorting of infected cells based on canonical markers of defined cell populations is the preferred approach for identifying phenotypic properties of infected cells; however, expansion of the PheP-Seq assay , may facilitate this process in the future.Reviewer #2 (Recommendations For The Authors):1. The authors have chosen to lump all R5 viruses together in terms of their entry phenotype, giving all viruses an equal chance of infecting all potentially susceptible cell types. This ignores the fact that normal HIV is selected to infect cells, requiring a high density of CD4 as is found on T cells. We use the term R5 T cell-tropic to describe "normal" HIV. The ability to efficiently enter cells that have a low density of CD4, such as macrophages and microglia, involves the evolution of a distinct phenotype, termed macrophage tropism . This happens most often in the CNS where T cells are infrequent thus potentiating evolution to infect an alternative cell type. This change in entry phenotype is dramatic and, like X4 viruses, results in phylogentically distinct lineages (PMID22007152). There are no sequence signatures for M-tropic viruses as there are for X4 viruses, but the fact that there are sequences shared between the CNS and lymphoid tissue makes it much more likely that there are T cells migrating around the body, including into the CNS, that are carrying R5 T cell-tropic virus with them, with the cells potentially clonally expanding in situ in the CNS. The persistence of a potential CNS T cell reservoir was the point we were trying to make in our recent paper (ref. 38), not only that these CSF rebound viruses were R5 viruses but they were selected for replication in T cells as seen by their dependence of a high density of CD4 for entry. This is the conclusion one would reach if clonally expanded viral sequences were shared between two lymphoid compartments. It is not necessary to ascribe properties of infection and clonal amplification to microglia cells when a more parsimonious explanation is that there are low levels of T cells in the CNS, especially in the absence of entry phenotype data showing these sequences encode an M-tropic entry phenotype. As is the authors are just adding to the unproven belief that virus in the CNS must be in myeloid cells, which in this case in particular we suspect is the wrong interpretation.We are impressed by reviewer 2’s recent work, suggesting the viral reservoir in the CNS may primarily consist of clonally-expanded R5 T-cell tropic viruses. We have adjusted our discussion to emphasize this possibility, and to highlight that viral entry phenotyping data will be informative for better understanding viral persistence in the brain.1. The authors noted that the frequency of intact proviruses is highest in the lymph nodes of 2/2 participants for which they had lymph node samples, relative to the other tissues examined. They thus conclude, "Together, these results indicate that intact HIV-1 proviruses are preferentially detected in lymphoid and gastrointestinal (GI) tissues." However, an examination of Figure 2 reveals that the total HIV copy number is highest in the lymph nodes of these two people. Thus, it doesn't seem like HIV is preferentially intact in the lymph nodes as much as they sampled more provirus from that tissue and therefore were able to detect more intact proviruses.We have adjusted our manuscript to indicate that the highest numbers of intact HIV-1 proviruses were present in lymph nodes, both in terms of absolute numbers and after normalization to the total numbers of cells analyzed.1. In Figure 1A, the legend should be changed so that "PMSC" is spelled out as "premature stop codon" for ease of reading. This is done for Figure 1B.We have corrected this issue as suggested by the reviewer.1. The pie charts in Figure 5 could be better labeled for ease of interpreting. In Figure 5C, instead of just labeling it as "P2" it could be "Distribution of CXCR4-using proviruses, P2", as an example. As it stands, it is hard to know what the figure is describing without reading the text.We have changed this accordingly.1. While all participants were taking antiretroviral therapy at the time of their death, they were not all suppressed when the tissues were collected. The authors are careful not to mention "suppressive ART" in the text, which is appreciated. However, the title should be changed to also reflect this fact.Thanks for pointing this out. From our perspective, ART is never fully suppressive, as low-level viremia  is detectable in almost all ART-treated persons. As such, it is not clear to us that “suppressive” necessarily implies suppression below the detection limits of commercial PCRs. We argue that ART can also be suppressive when plasma viral loads are in the range of 100 copies/ml. Nevertheless, we have changed the title to avoid confusion.Editorial comments:In addition to the reviewers suggestion, we feel that adding more information on how you define intact proviral sequence, e.g. are only disrupted essential genes or also in accessory genes considered? Previous studies have shown that brain-derived HIV-1 strains are usually CCR5-tropic, show high affinity for the CD4 receptor and frequently contain defective vpu genes. Some information and discussion if the brainderived sequences confirm these previous finding seems of significant interest.As described in our previous work , accessory genes are not considered in our definition of “genome intactness”; this is consistent with approaches other investigators have chosen . Within the genome intact sequences we identified in the CNS in our study persons, we found no evidence for deletions of vpu sequences; this has been emphasized in the revised manuscript.


Peri-implantitis is recognized as a complex pathology which could be defined as infectious inflammatory lesions that usually develop in the tissues around the implants. There are many protocols for the effective management of peri-implantitis that includemechanical debridement, the use of antiseptics and local/systemic antibiotics, and access and regenerative surgery formulated for the treatment of peri-implantitis. This study aims to evaluate the clinical outcomes of a mixed protocol for the regeneration ofdeep osseous defects. Records obtained from 27 patients who had already received treatment for peri-implantitis on one or more implants were retrospectively examined within the proposed time period between 24 and 30 months after their surgical treatment. Atotal of 33 implant sites were included and examined retrospectively. Descriptive statistics were calculated that include mean, SD, medians and confidence intervals at 95%. At the baseline, the mean Probing Depth was 8.19 ± 1.23 mm; Bleeding on Probing (BOP)was present on 29 out of 33 treated areas; pus was instead present on 17 out of 33 sites. At the time of final examinations, BOP was present on 9 out of 33 sites; pus was present only on two surgical sites. To conclude, a combined chemical-mechanical andregenerative decontamination therapy is effective in the treatment of peri-implantitis. Further investigation, which includes a control group and/or histologic findings, might be needed to ascertain the clinical results reported in the clinical studies. The currently emerging problem across the world is known to be peri-implant diseases which could be defined as infectious inflammatory lesions that usually develop in the tissues around the implants. -2Peri-iThe study was conducted after obtaining approval from the ethical committee. Records obtained from 27 patients who had already received treatment for peri-implantitis on one or more implants were retrospectively examined with the proposed period between 24and 30 months after their surgical treatment. A total of 33 implants were included and examined retrospectively. Patients were included as study participants if their records clearly showed that they had peri-implant bone defects on one or more implants whichhave been treated with the method detailed in this retrospective study. The records of those patients were considered excluded if there is any case of missing or incomplete pre-treatment, follow-up or final data, absent radiographs or surgical/regenerativeprotocols different from that described. Those patients who were treated non-surgically and those with Probing Depth (PD) of more than 6 mm and a lack of implant mobility were considered fit for surgical treatment. In order to be treated using regenerativesurgical procedures, patients must be non-smokers or smoke cigarettes that are fewer than 10 per day.10 All the patients were advised to receive a professional hygiene session 2-3 days prior to the surgical treatment, and this was performed to decrease theintraoral bacterial load. The proposed treatment is comprised of a combination of surgical and antimicrobial therapy. All patients were allocated to receive antibiotic prophylaxis with 2 g amoxycillin 1 hour prior to the surgery. Before the procedure, allpatients were advised to use 1 min mouth rinse with 0.2% chlorhexidine. Articaine 4% with 1:100,000 adrenaline was employed for the local anaesthesia by infiltration; a mandibular block was used in the mandible.A sharp, clean incision was made on the crest and vertical releasing incisions on the buccal side of mesial and distal sides. Once the muco-periosteal flap was elevated, the granulation tissue around the affected implant was then removed with the help ofsurgical curettes. Once the implant and the associated osseous defects were exposed, a Teflon insert was mounted on a piezoelectric surgicaldevice used to remove the debris and calculus from the implant's surface. Following this, chemical decontamination wasperformed using a 35% phosphoric acid gel. The gel was left in place for about 2 minutes, then suctioned and thoroughly washed with copious irrigation with saline solution. The flap was then allowed to release by sharp incision on the buccal side to have apassive adaptation, and it was sutured using monofilament sutures. A combination of vertical mattress sutures and single interrupted sutures were then allowed for the flap stability and closure. Patients were informed to keep an ice pack at 10 minutes intervalsand were advised not to brush on the treated area until 2 days after the removal of sutures. The post-op prescription mainly included: Amoxicillin + Clavulanic Acid 1 g tabs (twice a day) for 7 days, Metronidazole 250 mg tabs (thrice a day) for 7 days, Ibuprofen600 mg tabs (twice a day) for 3 days, and chlorhexidine mouth rinse 0.2% (twice a day) for 15 days. Supplements like vitamin B complex and vitamin D were also being prescribed. Follow-up exams were then scheduled every 2 months for the first 6 months and thenevery 6 months unless required.Descriptive statistics were calculated that include mean, SD, medians and confidence intervals at 95%. PD measurements at baseline level and one year at final follow-up were analysed using the one-way repeated measures ANOVA test, while the statisticalanalysis of the BoP and suppuration values at baseline and final evaluation was assessed using McNemar's test.All the baseline values have been briefed in the given table - 1. The study population consisted of 13 males and 14 females with a mean age of 53.8 when they received the surgical therapy. Nine patients were observed as smokers at the time of the surgicaltherapy, and 11 had a history of periodontitis. At the baseline, the mean Probing Depth was 8.19 ± 1.23 mm; Bleeding on Probing (BOP) was present on 29 out of 33 treated areas; pus was instead present on 9 out of 33 sites. At the time of surgicaltreatment, all patients were evaluated for ASA 1 and 2. They had received detailed written and oral information about the risks vs benefits of the treatment, and they had all given written informed consent. All patients had a confirmed diagnosis ofperi-implantitis based on the presence of Bleeding on Probing (BOP) and/or pus, localized oedema, erythematosus peri-implant mucosa and radiographic signs of bone loss. Twenty-seven patients who had been surgically treated for peri-implantitis affecting 33implants and followed for a minimum of 24 months were included in this retrospective evaluation; the mean follow-up time was 28.9 months (a range of 24-38 months). Post hoc tests using the Bonferroni correction revealed that the peri-implantitis treatmentelicited a significant reduction in PD from the baseline to the one-year follow-up , which was significant (p < 0.0005). At the time of final examinations, BOP was present on 9 out of 33 sites;pus was present only on two surgical sites. An exact Mc-Nemar’s test determined a statistically significant difference in the proportion of implants with BoP and pus pre and post intervention .The retrospective analysis in this study, 27 patients was managed with a mixed protocol based on the mechanical debridement, chemical/pharmacological decontamination and bone regeneration. The mean probing depth went from the initial 8.19 mm to 4.21 mm. Oneimplant in the region of #18 had a negative outcome because the clinical conditions have considerably worsened with time and returned to the initial conditions. Moreover, all the other implants had noticeable improvement compared to the baseline. The PDreduction obtained as the results are already at 12 months and also at the final follow-up are satisfactory in terms of containment of the progression of the disease. Data relating to Bleeding on Probing and pus might confirm the reduction of inflammation andinfection. Six patients developed some relapse of PD, but only in one of those with the probing depth observed severe (more than 8 mm), with the simultaneous presence of bleeding and pus was reported. In the remaining cases, the relapse was observed to beminimal (1 mm), with probing depth in all was ranged between 4 mm and 5 mm and the residual BOP is noticed in only one case.The causes of failure could be estimated as many, from the patient’s non-compliance to the incomplete surface decontamination (where their complete sterility is neither predictable nor verifiable).  MoreoverA combined chemical, mechanical and regenerative decontamination therapy is effective in the treatment of peri-implantitis. Further investigation, including a control group and/or histologic findings, might be needed to ascertain the clinical results reportedin the clinical studies.