P < 0.05). The correlation coefficient between the different ranges of testicular volume and the incidence of varicoceles was 0.829. Increased testicular volume may be also a factor for the development of varicoceles. Dilated peritesticular vessels may be collateral veins of spermatic veins, anterior and posterior scrotal veins, or proximal vas deferens.The purpose of this study was to curate clustered findings of duplex ultrasound in the evaluation of spermatic venous varicoceles, and deliver more responses to the present concerns. Archives of 979 men who had undergone scrotum and spermatic venous plexus duplex ultrasound were reviewed. In the duplex ultrasound interrogation, the sizes of the larger vessels of the spermatic venous plexus, peritesticular vessels, and testicular volume and relevant parameters were measured. Findings of the vessels were analyzed. One hundred and eight-one out of 979 patients had varicoceles. Color Doppler flow signal was rendered in veins of pampiniform plexus but not in peritesticular vessels in 501 out of 979 patients; 101 out of 501 patients had veins of pampiniform plexus ≤ 3 mm, no color Doppler flow signal could be rendered in the veins in the 101 patients at supine and standing positions without Valsalva maneuver, color Doppler flow signal could be rendered in the veins in 82 out of 101 patients at supine and standing positions with Valsalva maneuver; no color Doppler flow signal could be rendered in the veins from 19 out of 101 patients with and without Valsalva maneuver at supine and standing positions. 37 out of 979 patients with 61 ipsilateral testicular volume ≤ 5 mL had no vessel diameter > 2 mm. The incidences of varicoceles corresponding to different ranges of testicular volume of 1–5 mL, 5.1–10 mL, 10.1–15 mL, 15.1–20 mL, 20.1–25 mL, and 25.1–30 mL were 0.0%, 6.9%, 8.3%, 6.63%, 20.94%, and 59.1%, respectively. The comparisons of incidences of varicocele between distribution percentages of different ranges of testicular volume of 1–5 mL and others (of 5.1 mL and more) were all significant (all  Of varicocele, there has been an estimated prevalence of 15% in the general population, 35%-39% in primary male infertility3. Diagnosis of varicoceles depends on clinical scenarios, mainly the presence of dilated and tortuous veins in the spermatic cord, and medical imaging evaluation. In accordance to ultrasound evaluation of varicoceles: guidelines and recommendations of the European Society of Urogenital Radiology Scrotal and Penile Imaging Working Group (ESUR-SPIWG) for detection, classification, and grading, varicoceles is defined by the presence of multiple veins greater than 3.0 mm with concomitant retrograde blood5. Copious studies on diagnosis, grading, mechanism, and management of varicoceles have been published, many consensuses have achieved, however, some controversies and concerns remain unsolved10. The purpose of this study was to curate clustered findings of duplex ultrasound in the evaluation of the scrotum and spermatic venous varicoceles, and deliver more response to the present concerns.Varicocele is an abnormal dilation of the pampiniform plexus of veins within the inguinal canal and scrotum, and it is a relatively common clinical problem, both in asymptomatic men and even more prevalent in subfertile men, representing the most common potentially treatable cause of male infertilityArchives of 1976 men who had undergone consecutively scrotal duplex ultrasound evaluation in a tertiary hospital from January 1, 2016 to January 1, 2020 were reviewed. 979 men who had undergone scrotum and spermatic venous plexus duplex ultrasound for a clinically visualized venous plexus or collateral vein dilatation, palpable venous plexus enlargement, or spermatic cord fullness, which increased in size on assumption of the upright position or during the Valsalva maneuver, or other causes were included, and 997 patients who had spermatic cord hydrocele, hydrocele of tunica vaginalis testis, inguinal hernias, crytorchidism, testicular tumors, testicular torsion, epididymical lesions, epididymal agenesis, with history of varicocele repair, and who had not undergone spermatic venous plexus duplex ultrasound were excluded, as shown in Fig. The ultrasound systems used during this period included Hitachi-Aloka prosoundα7 ; Mindray DC-8 ; Mindray Resona 7 ; Siemens Acuson S2000 ; Philips EPIQ5 ; GE Logiq E9 . The patients were examined in the supine and standing position consecutively in the air-conditioned rooms with an ambient temperature of 24–30 °C. After the scrotum was exposed properly, adequate volume couple gel (heated to 33–35 °C) was placed onto the scrotum. The transducer was placed on the scrotum without exerting pressure. The linear-array transducers equipped in different ultrasound imaging systems with frequencies from 5 to 14 MHz were used to scan the scrotum, and multiple parameters were assessed, including bilateral testicular volume, echogenicity, echotexture, anatomic variants, abnormalities of the testes, epididymides, spermatic venous plexus vessels, peritesticular vessels, tunica vaginalis, inguinal canal, and scrotal wall. Color Doppler flow imaging and pulsed wave Doppler assessment were performed as an integral part of examination to detect and characterize venous reflux at the level of the inguinal canal, supratesticular region, and level of the testis. The sizes of the larger vessels were measured, irrespective of vessel location , patient position (supine or standing), or with and without Valsalva maneuver. In patients with an isolated right-sided varicocele, the abdomen was examined for abdominal and retroperitoneal pathology, as well as congenital vascular anomalies. For each examination, the examination mode was preset to “small parts and testis”, parameters of gain, depth gain compensate, focus, depth, and scale or pulse repetition frequency were adjusted to an adequate status. Images of duplex ultrasound imaging with and without measurements were saved in the PACS. The duplex ultrasound findings of spermatic venous plexus vessels, peritesticular vessels, epididymis, testis, and scrotum were analyzed.4. The incidences of varicoceles corresponding to different ranges of testicular volume of 1–5 mL, 5.1–10 mL, 10.1–15 mL, 15.1–20 mL, 20.1–25 mL, and 25.1–30 mL were calculated using percentile method. The incidences of varicocele between distribution percentages of testicular volume of 1–5 mL and others (of 5.1 mL and more) were compared using Chi-square analysis. Nonparametric correlations (spearman) were used to evaluate the relationship between the different ranges of testicular volume and the incidences of varicoceles. The level of statistical significance was set at p < 0.05 (2-tailed). Statistical analyses were performed with SPSS 20  statistical software.The testicular sizes were all re-calculated using up-to-date formula volume = Length × Height × Width × 0.71All procedures followed were in accordance with the ethical standards of the responsible committee of the first affiliated hospital of Hainan medical university on human experimentation  and with the World Medical Association Declaration of Helsinki (revised in 2000). The study was approved by the institutional review board of the first affiliated hospital of Hainan medical university, and informed consent was waived due to the retrospective design.P < 0.05). The correlation coefficient between the different ranges of testicular volume and the incidence of varicoceles was 0.829 (P = 0.042). Distributions of testicular volumes, maximal venous diameters of pampiniform plexus and percentiles of varicoceles were listed in Table One hundred and eight-one out of 979 patients had spermatic venous diameter > 3.0 mm. Of these dilated veins, 144 out of 181 veins located left side, nine out of 181 veins located right side, and 28 out of 181 veins located bilaterally. Color Doppler flow signal and reflux flow could be detected using duplex ultrasound in these veins, as shown in Fig. 7 . Other causes include that the incompetence of venous valves results in reflux of venous blood and increases hydrostatic pressure; "nutcracker" phenomenon that the proximal left renal vein decreases with decrease in the aortomesenteric distance and angle; renal tumor compression, and so on9. In addition, we believe that the common presence of anatomical variations of testicular vessels and relevant vessels may be also the cause of varicoceles15. For if the distribution of testicular drainage in different veins is not appropriate, the spermatic vein drainage and hemodynamics may be affected, and these may cause left side, or right side varicoceles, or bilateral varicoceles.In our series of 979 patients undergoing scrotal evaluation, 181 patients with varicoceles (≥ 3 mm) had not referred to physician before, accounting for 18.5% (181/979) of all evaluated patients. The reason is that majority of them had mild symptoms, and they did not pay much attention to the symptoms and / or were not aware of the underlying disease. The main cause and mechanism of varicoceles of pampiniform plexus has been interpreted as that the right testicular vein usually drains into the inferior vena cava while the left testicular vein drains into the left renal vein and gets confluence with the renal venous flow, then drains into the inferior vena cava. This anatomical difference induces relatively weak hemodynamics in the left testicular vein, which accounts for the predominance of left sided varicoceleP < 0.05), and the relationship between the different ranges of testicular volumes and the incidences of varicoceles was strong (correlation coefficient of 0.829). These indicate that the volume of testicular drainage associates with the testicular volume, the larger the testicular volume the more the drainage takes place. If the drainage does not match the arterial perfusion exactly, it may develop blood stasis and result in varicoceles.In our study, no ipsilateral varicocele occurred in 37 adult patients with 61 ipsilateral testicular volume < 5 mL , and no diameter of pampiniform plexus was < 2.0 mm in patient with ipsilateral testicular volume > 20 mL. The comparison of incidences of varicocele between distribution percentages of different ranges of testicular volume of 1–5 mL and others (of 5.1 mL and more) were all significant  could be rendered in normal size veins of pampiniform plexus and some small vessels (diameter ≤ 2 mm) of pampiniform plexus and peritesticular veins. These indicate that flow reflux presents commonly in veins with dilated diameter, and also presents in veins with normal diameter, while veins with diameter > 3 mm (varicoceles) are almost definitely abnormal. So we believe that dilated venous diameter is the fundamental parameter of varicoceles, and flow reflux is the second; although the duration of reflux time at Valsalva maneuver, an important parameter of varicoceles, can be used for varicoceles grading18. Tubular ectasia of the rete testis is a rare vessel entity that can be encountered occasionally in the scrotal evaluation19. Tubular ectasia of the rete testis was found in two of the 181 patients with varicoceles in this study, and no color Doppler flow signal was rendered from them. Tubular ectasia of the rete testis occurs in the testis, which is different from intratesticular varicoceles, and it does not subject to the dilated vein of pampiniform plexus.There are 630 out of 979 patients with diameter of scrotal vessel ≤ 3 mm in this series. The profile of dilated scrotal peritesticular vessels may be either subclinical varicoceles according to the present diagnostic criteria of varicoceles, or collateral vein dilatation or other entities. Proximal vas deferens can be traced originating from the epididymis, the wall of it is thicker than vein, and it is recognizable (as shown on Fig. The potential limitations of the present study were that the assessment and measurement were performed by different physicians on different ultrasound imaging systems, the interobserver agreement had not been evaluated, which might cause errors in some extent. However, our previous studies and experiences had shown that the measurements of vessels on high frequency ultrasonography and at zoom mode are reproducible, which causes only a little error, so it may be overlooked. The other caveats are that the drainage route of spermatic veins had not been tracked, and the specific causes and mechanism for varicoceles were not validated and further understood.Spermatic venous varicoceles and dilated peritesticular vessels present various manifestations at duplex ultrasound imaging interrogation. The relationship between the different ranges of testicular volume and the incidences of varicoceles was strong, and varicoceles had not found in spermatic veins of ipsilateral testicular volume ≤ 5 mL, which indicate that increased testicular volume may be also a factor for the development of varicoceles. Venous diameter > 3.0 mm is the fundamental criterium of varicoceles, and flow reflux is the second. The dilated peritesticular vessels include collateral veins of spermatic cord veins, anterior and posterior scrotal veins, or proximal vas deferens .Supplementary Legends.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6A.Supplementary Figure S6B.


The inflammatory diseases rheumatoid arthritis (RA) and periodontitis show similarities in misbalances of cytokine levels, such as tumor necrosis factor-α (TNF-α). RA has been treated for two decades with TNF inhibitors which are effective by blocking TNF’s destructive action. Since RA and periodontitis show similarities in high levels of TNF, the periodontal status of RA patients may improve with the use of anti-TNF therapy. To assess this, a systematic review with special emphasis on duration of therapy was performed to evaluate the effect of anti-TNF-α treatment on the periodontal status of RA patients. Overall, studies showed an improvement in periodontal health with anti-TNF therapy. When analyzed over time (6 weeks to 9 months), it became apparent that initial improvements concerned bleeding on probing (BOP) and gingival index (GI) after therapy duration of 6 weeks. Periodontitis parameters that improved after prolonged treatment were: probing pocket depth (PPD) after 3 months and clinical attachment level (CAL) after 6 months. In conclusion, this systematic review reveals that anti-TNF treatment is therefore not only beneficial for rheumatic joints but also for the gums of rheumatoid arthritis patients. We propose that the sequential tissue recovery due to anti-TNF therapy progresses as follows: 1. block of diapedesis by lowering vessel permeability, 2 fewer leukocytes in the inflamed tissue, and 3. reduced proteolytic activity and subsequent repair of collagen fiber functionality and normalization of osteoclast activity. Clinically, this could lead to a decrease in bleeding on probing and ultimately in an improved clinical attachment level. Both periodontitis and rheumatoid arthritis are inflammatory diseases. Periodontitis is caused by an inflammatory response to microbes and bacterial toxins, eventually leading to destruction of supporting periodontal tissues around the teeth . RheumatTumor necrosis factor alpha  is a pro-inflammatory cytokine produced in occurrence of inflammation by cells like macrophages and monocytes . It is aTNF inhibitors are used clinically to counterbalance the high TNF levels accounting for joint inflammation, hereby preventing TNF tissue damage in RA. TNF inhibitors have been available for inflammatory diseases like rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, ulcerative colitis and Crohn’s disease since 1998. The presently available TNF inhibitors are infliximab, adalimumab, golimumab and etanercept and certolizumab pegol. These inhibitors have an immunoglobin (Ig) structure in common. Infliximab has a 65% similarity to human IgGs, and golimumab and adalimumab have the highest similarity with human IgG. Etanercept is a recombinant fusion drug existing of TNF p75 receptor and the fc component of human IgG1. Anti-TNF reduces inflammatory reaction of the body by blocking TNF-α, hence preventing it to bind on its receptor (TNFR1 or TNFR2). However a prolonged use of TNF inhibitors has a few possible side effects like: hepatotoxicity, malignity, greater risk for infection, immunogenicity and cutaneous reactions.Heart failure is a contraindication for anti-TNF therapy . High leIn vitro and in vivo studies have already proven that the use of anti-TNF (infliximab and etanercept) inhibits osteoclast formation , 19, 20.Previous work summarizing the effectiveness of anti-TNF therapy in RA and ankylosing spondylitis (AS) has suggested a a positive spin-off for the periodontium , 21, 22.For the literature search a methodological approach was adopted by using PRISMA . This method was used to reduce bias in literature selection by the authors.The electronic search was carried out in the following databases: Embase, Pubmed and Web of Science. Studies up to January 2020 and publications in English were included in this search. The keywords used for the search were: “periodontitis,” “periodontal diseases,” “periodontal,” “adalimumab”, “etanercept”, “infliximab”, “certolizumab pegol”, “antibodies tumor necrosis factor- alpha,” and “TNF-alpha inhibitors” Table 1,To select suitable publications a set of inclusion and exclusion criteria was used. All in vitro and animal studies were excluded. Cohort studies, case reports, randomized controlled trials, and longitudinal studies in English were included. Furthermore, only studies that tested at least one of the clinical periodontal parameters  or radiographic parameters were included. Outcomes of clinical trials compared the effect of anti-catabolic medication on positive or negative effects on the periodontium. Articles that did not meet this outcome were excluded.All titles and abstracts of the publications from the electronic search were screened by two reviewers (FZ and TV) and discussed. When mutual agreement between the two reviewers could not be reached or when the suitability of the publication was questionable the full article was examined by both. The selected literature was divided into the different follow-up periods and presented in tables summarizing the effect of the medications on the following periodontal parameters: plaque index (PI), gingival index (GI), BOP, PPD, and CAL. PI is a score where supragingival plaque is recorded on four or six sites of each tooth. The score defines the absence or presence of plaque by marking it with a positive or negative score. Accumulation of these scores finally results in the PI, which varies from 0 till 100%. The GI can be used to evaluate the condition of the gingiva and is scored at four sites around the teeth. The score varies from 0 to 3, a score of 0 for a normal gingiva and 3 for a severe gingival inflammation . BOP is The electronic research that was carried out resulted in a total of 1,571 articles. After manually removing the duplicates a total of 1,209 publications remained. An additional 1 record was found through manually screening reference lists of articles that were found. All the 1210 articles were manually screened for suitability by reading the titles and abstracts. This resulted in a full text read of 23 articles of which 10 publications were excluded with reasons like: no full text available, no clear description of the used materials and methods, or use of new medication during follow-up period. Finally, 13 publications were included in this review Figure 1The first group of studies that will be discussed is the group with no follow-up assessment which consisted of 3 studies Table 2.The following results describe studies on TNF inhibitors used for 30 days, 6 weeks, 3 months, 6 months and 9 months. Results are described and compared to baseline measurements and potentially give insight in which parameter improves over time.Üstün et al. carried out a longitudinal study of 30 days assessing the effect of infliximab and adalimumab in RA patients with periodontitis. 30 Days of anti-TNF-α therapy increased GI significantly while PI, BOP, PPD, and CAL did not change significantly over this time Table 2.Even in RA patients without periodontitis anti-TNF resulted in an increase of GI .Studies with a follow-up period of 3 months are presented in After 6 months Table 2 Though not all parameters were analyzed in all studies it is apparent that significant improvements have been reported for all parameters. Savioli et al. tested the effect of the use of TNF inhibitors  in RA patients with and without periodontitis. In both groups there was no significant improvement in BOP, PPD or CAL. However in the RA group with periodontitis the PI decreased significantly (p < 0.03) . Fabri eThe final study with a follow up period of 9 months is Pers et al. Table 2.In order to control for the efficacy of anti-TNF for the original application, to minimize the rheumatoid arthritis induced inflammation burden, various parameters can be measured. Nearly all studies, except Pers et al. measured the Disease Activity Score 28 (DAS28)  and cytoBy evaluating changes in these parameters, the effectiveness of TNF-inhibitors in reducing inflammation can be evaluated. These findings are shown in Most of the studies showed a decrease in DAS28, serum CRP levels or TNF levels and thus the anti-inflammatory effect of anti-TNF therapy. A few studies did not show a decrease in these inflammatory parameters. Schiefelbein et al. showed no decrease in serum CRP levels when RA patients on anti-TNF were compared to RA patients without anti-TNF . Ortiz eThis literature review presents an overview of the possibly beneficiary effect of TNF inhibitors used in rheumatoid arthritis on periodontal parameters, with special emphasis on which parameters improve over time. The effectiveness of TNF inhibitors in periodontal therapy can be assessed when measuring the anti-inflammatory effects of TNF inhibitors on periodontal parameters of the examined subjects. Measurement of DAS28 is essential in all studies in order to establish effect on joints due to TNF inhibitors in patients. Studies have shown that it takes 2 weeks of anti-TNF  administration to observe a clinical response in RA and AS patients –41. TherThe time point 6 months is the most widely studied, therefore, we summarized only this time point quantitatively Figure 23 proved that long-term administration (>12 months) still had a positive effect on cytokine levels, and periodontal parameters. Mayer at al. in 2009  . Seconda in 2009  and in 2 in 2009  also proAs earlier described responsiveness to the anti-TNF therapy has been evaluated per study group in the included studies and not per individual. Lack of responsiveness to anti-TNF therapy might be correlated to ineffectiveness of improving periodontal parameters. Therefore, it is of importance to evaluate if lack of responsiveness in one subject translates in the same subject to a lack of improvement in periodontal parameters. When identifying predictive parameters, anti-TNF therapy could be applied in therapies combating inflammatory diseases. Alternatively, in cases of non-responsiveness, IL-6-R inhibitor treatment could be an option, as described by Kobayashi and coworkers . ShortcoPers et al.  showed aBased on the findings described in this paper and based on general cell biological knowledge of TNF-α , we propThis overview presented promising results that anti-TNF therapy is beneficial in the treatment of both RA and periodontitis by improving periodontal parameters of RA patients. One would like to be able to predict which patients with both RA and periodontitis would benefit from which TNF inhibitor. Future research is needed to elucidate this further.Idea for the systematic review: TV. Literature search and first draft: FZ. Writing: FZ and TV. Completion: FZ and TV. All authors contributed to the article and approved the submitted version.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.


Virus‐like particles (VLPs) are ideal platforms for such 3D vaccines, as they allow the display of complex and native antigens in a highly repetitive form on their surface and can easily reach lymphoid organs in intact form for optimal activation of B and T cells. Adjusting size and zeta potential may allow investigators to further fine‐tune delivery to lymphoid organs. An additional way to alter vaccine transfer to lymph nodes and spleen may be the formulation with micron‐sized adjuvants that creates a local depot and results in a slow release of antigen and adjuvant. Ideally, the adjuvant in addition stimulates the innate immune system. The dynamics of the immune response may be further enhanced by inclusion of Toll‐like receptor ligands, which many VLPs naturally package. Hence, considering the 3Ds in vaccine development may allow for enhancement of their attributes to tackle complex diseases, not usually amenable to conventional vaccine strategies.Vaccines need to be rationally  However, the vaccine proved less efficacious against the most severe pneumonia form of plague.The practice of variolation, also called “inoculation or insertion,” was used in China in the 10th century to immunize people against smallpox. Live, virulent viruses were collected from scabs of infected victims and inserted into the skin of healthy individuals. The concept of variolation against smallpox spread further to India in the 17th century followed to southwest Asia and BalkansThe Salk vaccine “inactivated poliovirus” was developed by Jonas Salk in the 1950s, while the Sabin vaccine “live attenuated poliovirus” was advanced by Albert Sabin for oral administration. Though highly efficacious, the Sabin vaccine is only used rarely nowadays due to the potential to revert to a virulent form or cause disease in immunocompromised individuals. Mass immunization against poliovirus resulted in the eradication of the disease in most parts of the world.Taken together, traditional vaccines (inactivated or attenuated pathogens or toxins) have shown high immunogenicity and ability to stimulate the innate and adaptive immune system conferring long‐term memory and protection. Such high immunogenicity, particularly where viral vaccines are concerned, is due to several factors such as the ability of the pathogen to replicate, the repetitive surface geometry of the virus and its particulate nature.design, delivery, and dynamics.The 21st century has witnessed an advancement in molecular genetics, microbiology, immunology, and genomics techniques in applied vaccinology. Scientists have taken advantage of viruses by making them invaluable sources for generating safe and novel nanoscale scaffolds termed virus‐like particles (VLPs). VLPs are multiprotein structures that resemble viruses in many characteristics.22.1Crick and Watson have stated in 1956 that “it is a striking fact that almost all small viruses are rods or spheres.” “These shells are constructed from a large number of identical protein molecules, of small or moderate size, packed together in a regular manner”.The majority of viruses have a coat or a nucleocapsid consisting of multiple copies of single or just a few different kinds of structural proteins arranged in icosahedral or helical shapes. Coat proteins (CPs) can self‐assemble in artificial expression systems or in vitro spontaneously with or without the aid of nucleic acids (NA) or scaffold proteins. According to purely mathematical principles, an icosahedron is built from 60 identical units arranged in 5‐3‐2 symmetry which is used to describe the rotation possibilities of an icosahedron around an axis.T) of the coat is determined by two integers h and k and is defined as T = h2+hk + k2.h and k correspond to the number of steps, which must be traveled along two axes in a hexagonal grid across the centers of pentameric and hexameric subunits in order to reach one vertex of icosahedron from another. Figure T = 1 coat symmetry using porcine circovirus serotype 2 (PCV2)   Figure  and T = ) Figure . HepatitT = 1 particle with 60 spikes. Due to several reasons listed in Table T = 1 VLPs may often be not ideal for a vaccine platform and T = 3 particles may be more favorable.It is interesting to note that most artificially designed synthetic VLPs consisting of a trimerizing and pentamerizing alpha‐helical domain form T = 3 configuration are ssRNA bacteriophages such as MS2, AP205, and Qβ as well as several plant viruses such as cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CuMV). There is an interesting difference between the ssRNA bacteriophages and ssRNA plant viruses. First, the folds of the CPs are entirely different in both cases, CPs of plant viruses have classical jelly‐roll β‐barrel fold,H cell epitope in a given protein is proportional to the number of amino acids (a.a.) present in the protein. VLPs derived from plant viruses have a longer epitope sequence and therefore may be superior to bacteriophage VLPs in activating TH cells even though both have T = 3 symmetry.Examples of VLPs with subunits arranged in P = μ × ρ where P refers to the pitch of the helix, μ is the number of structural units per turn of the helix, and ρ is the axial rise per subunit. A well‐known example of helical rod‐shaped VLPs with hollow central channel is the tobacco mosaic virus–like particles.In general, icosahedron architecture is more prevalent than the helical shape. The definition of helical symmetry covers a broad area of geometries such as stacked rings, rods, or spring‐shaped coils.T = 3 particles and the above‐mentioned rod‐like prolate icosahedrons, but also into T = 1T = 4 particles,Remarkably, the CPs of RNA phages can self‐assemble not only into the classical 2.2TT‐VLPs.TT‐VLPs is a T = 3 VLP identical to the parent CuMV‐VLPs where subunits A are arranged in pentamers of five‐fold symmetry and three dimers of subunits B and C are arranged in hexamers in three‐fold symmetryBioorganic chemistry offers different artificial ways to modify proteins and other molecules. For successful chemical transformation of the biological molecules in VLP‐based vaccine development, several aspects should be considered: (a) The reaction should be efficient in an aqueous solvent with physiological pH values; (b) low concentrations of non‐toxic reagents should be used; and (c) the tertiary and quaternary structure of the antigen and VLP should be preserved. Using different chemical modification strategies, we have reached numerous proofs of concept in humans and animals. For example, vaccination with Qβ‐VLPs displaying angiotensin II on the surface resulted in reduced blood pressure.H cells and B cells under normal as well as more challenging conditions such as those found in aged patients. This is due to the fact that preexisting immunity to the chosen TT epitope is very broad in humans  as the peptide binds to essentially all human leukocyte antigen‐DR isotype molecules and everybody has been immunized many times against TT.T = 3 icosahedron geometry and without exposing the peptide on the VLP surface, avoiding interference of TT‐specific antibodies  It is adaptable to clinical applications; (b) CuMVTT‐VLP monomers have a size of 24 kDa, which can serve as an additional source of  TH cell epitope; (c) the VLPs incorporate RNA derived from the E coli production strain serving as a Toll‐like receptor (TLR)7/8 agonist; (d) the VLPs are stable for long time periods at 4°C and even 20°C; and (e) the platform is safe and highly immunogenic in mice, rabbits, dogs, cats, and horses and therefore is also expected to offer a good safety profile and immunogenicity in humans.The newly developed platform is thought to enhance the interaction between Ts Figure . This haTT platform, we have generated a proof of concept for different preclinical vaccines as listed in Table TT‐VLPs, which induced neutralizing antibodies without enhancing Dengue virus infection.TT‐VLPs to treat chronic itch in dogs. Several VLP‐based vaccines in preclinical and clinical studies are based on these simple chemical methods.TT‐VLPs or Qβ‐VLPs. The method showed efficacy and induced specific T cell response protective against tumor progression in an aggressive transplanted melanoma murine model.Using this new CuMVE coli. Yields of such VLPs are high, and the cost of goods is in orders of magnitude lower than conventional production.Many antibacterial vaccines are based on conjugation of carbohydrates to carrier proteins. VLPs are obvious candidates for such carrier proteins as they enhance the immunogenicity of conjugated antigens greatly.2.3E. coli is the most widely used system for the production of non‐enveloped VLPs. VLPs self‐assemble on prokaryotic RNA (pRNA) during the expression process.TT‐VLPs can successfully be expressed in E coli host. Cryo‐electron microscopy analysis of the VLPs demonstrates additional electron density in its interior cavity. This ordered structure most likely reflects the tightly bound host pRNA suggesting its role in stabilizing the subunits of the VLPs.VLPs can assemble on a polyvalent scaffold of NA, usually RNA. Generally, the CP of viruses play a role in organizing the packaged NA; conversely, NA plays a role in structure's assembly and immunogenicity of VLPs. A highly ordered NA structure can be seen in bean pod mottle virus,A recent study performed by us has used the disassembly/reassembly method to compare three different types of RNA packaged inside Qβ‐VLPs displaying the M2 ectodomain from influenza virus as an antigen. pRNA, eukaryotic RNA (eRNA), or tRNA has been used, and the results indicated that the type of packaged RNA can alter the induced protective humoral immune response in a murine influenza model. Specifically, pRNA packaged in Qβ‐VLPs induced the most protective IgG2b/c, while eRNA showed more shift to IgG1 with less protection. The study provided evidence that the shifting of the Ab response is dependent on TLR7 and it rules out the role of the cytoplasmic mitochondrial antiviral signaling protein (MAVS) and retinoic acid–inducible gene‐like receptor (RIG‐1). Such findings are crucial for rational design of VLP‐based vaccine aiming at inducing the most desirable and strong B cell response.E coli cells.The second method is the enzymatic digestion method, which is achieved by treating the VLPs with RNase to remove the RNA. Subsequently, the empty coat VLP can be repackaged with small oligonucleotides of choice, yielding a loaded VLP. This method has been used in different studies utilizing the bacteriophage Qβ or HBcAg.33.1The protection conferred by a VLP‐based vaccine is essentially dependent on the induced humoral and/or cellular immune response. However, the science of vaccine administration route is a poorly developed and described area. This is mainly due to the fact that vaccine trials lack standardized comparison of the injection site, needle length, or injection techniques.™) use IM route of administration as shown in Table Evidence‐based medicine aims at improving the quality of health methodologies. Thorough careful assessment of published clinical data regarding the route of vaccine administration subcutaneous (SC) vs intramuscular (IM) revealed that the current practice is based on tradition rather than clinical data.When comparing SC to IM routes of vaccine injection, IM has shown a faster rate of absorption of administered materials.Under normal non‐inflamed conditions, very few resident immune cells such as APCs are found in muscles.There is evidence that intradermal vaccination may be superior to other routes of immunization. Indeed, a study published in 2009 has used a near‐infrared fluorescent dye (NIR) coupled to simian‐human immunodeficiency virus (SHIV)–like particles to track their movement in SKH‐1 mice using different immunization routes. The study has observed that intradermal immunization with SHIV‐VLPs involved a broader range of lymph nodes (LNs). This resulted in the production of the highest Ab titers confirmed by the increase in germinal centers in the spleen as well as the highest antigen‐specific cytotoxic T‐lymphocyte (CTL) response.With the advent of oncolytic viruses injected directly into the tumor, this route is also investigated for VLPs. The bacteriophage Qβ‐VLPs filled with A‐type CpGs  have been used for intratumoral injections and are currently being examined in combination with systemic anti‐PD‐1 checkpoint inhibition in two different clinical trials for patients with advanced melanoma who showed resistance to anti‐PD‐1 treatment. The recently collected data from the ongoing trials show manageable toxicity and potential to reverse the resistance to anti‐PD‐1 (NCT02680184/NCT03084640). Lizotte et al have investigated a further route of application and shown that inhalation of cowpea mosaic virus (CPMV)–like particles caused regression of an established B16F10 lung melanoma by generating an efficient systemic anti‐tumor immune response. The study has shown that an important player of the generated anti‐tumor response in the tumor microenvironment was neutrophils which could rapidly take up CPMV‐VLPs.A combination of different routes has also been investigated using human immunodeficiency virus (HIV)–like particles which resemble the virus envelope protein in its native conformation. It has been shown that that priming with HIV‐VLPs intranasally followed by sub‐cheek route for boosting resulted in the highest titers of specific immunoglubulin B (IgG) compared to the other combination of priming and boosting routes used .H cell–dependent, both IgM and IgA antibodies are TH cell–independent.+ and CD4+ T cells.Mucosal Ab responses may be an interesting case where the route of immunization is concerned. SC immunization of mice with RNA‐loaded VLPs leads to strong systemic immunoglobulin M (IgM), immunoglobulin A (IgA), and IgG responses. While the IgG response is TIn conclusion, it seems that VLPs efficiently reach lymphoid organs from most injection sites and prime similar but not always identical immune responses.4As discussed above for the delivery, rules for induction of Ab vs T cell responses may differ and both design and delivery of VLPs have important consequences for the dynamics of the immune response.4.1VLP‐based vaccines are made of a restricted number of antigens or individual components of the targeted pathogen, hopefully able to confer a protective and/or a therapeutic effect. Accordingly, the protective epitope must be displayed in its native form, at least if the induction of Abs is the desired response. This may be different for T cell‐based vaccines, where native display of the epitope is not required. Furthermore, VLP‐based vaccines must be optimized for antigen density, dose, and prime/boost interval to obtain a potent Ab response. Unexpectedly, a recent study has demonstrated that such criteria are not necessary for achieving high‐avidity T cell responses.4.2B cell responses are optimally stimulated by repetitive particles displaying native epitopes and which are associated with a potent adjuvant for B cells. VLPs loaded with RNA or CpGs and displaying full‐length antigens on their surface are therefore ideal for induction of Ab responses. Depot formation may be less important for vaccines aiming to induce Abs, since VLPs are recognized by natural IgM antibodies which recruit C1q and lead to deposition on follicular DCs (FDCs).Conventionally, vaccines aim to induce long‐lived Ab responses rather than memory B cells.4.3H 1 as well as CTL responses despite the fact that they do not carry any genetic material for replication.+ and CD4+ T cells, respectively.b gp33 epitope. The most striking observation involved CCL2 chemokine which was distinctly expressed in DCs 24 hours following immunization with Qβ(type‐B CpGs)‐p33, a potent TLR9 ligand. The identified pathway is thought to play an important role in DCs activation and subsequent induction of potent (CTL) response.A VLP‐based vaccine displaying T cell epitopes can elicit a strong T+ and CD8+ T cell response in a HPV mouse model. These findings indicate that physical linkage of both antigen and adjuvant in a VLP‐based vaccine may not be necessary for effective T cell activation. In contrast, simple, admixing VLPs did not enhance B cell responses, indicating the rules for T and B cell responses are different . CuMVTT‐VLPs are nanoparticles with packaged RNA as TLR7/8 agonist while MCT is a micron‐sized depot‐forming adjuvant capable of activating the inflammasome. Such formulation has increased the resultant specific CTL response in a murine melanoma model.We have recently harnessed the physiological properties of the lymphatic system by formulating CuMV5The 3Ds of vaccinology, design, delivery, and dynamics, are 3 key components for the efficient generation of a protective vaccine. Optimal design allows repetitive display of native antigens on VLPs loaded with TLR ligands for direct stimulation of B cells for IgG and IgA production as well as enhanced T cell responses. Optimal delivery may be key for inducing mucosal responses, or enabling dose sparing and mediating direct oncolytic activity if applied into the tumor. Optimally designed and delivered vaccines will result in optimally dynamic immune responses, with the desired outcome of strong T cell responses, highly specific Ab responses, and optimal clinical efficacy.The authors declare no conflict of interest.Video S1Click here for additional data file.


Here, we generated and analyzed cFLIP epidermal KO mice with additional TNF deficiency. Intriguingly, the ablation of TNF rescued the pathological phenotype of epidermal cFLIP KO from characteristic weight loss and increased mortality. Moreover, the lack of TNF in these animals strongly reduced and delayed the epidermal hyperkeratosis and the increased apoptosis in keratinocytes. Our data demonstrate that TNF signaling in cFLIP-deficient keratinocytes is the critical factor for the regulation of skin inflammation via modulated cytokine and chemokine expression and, thus, the attraction of immune cells. Our data suggest that autocrine TNF loop activation upon cFLIP deletion is dispensable for T cells, but is critical for neutrophil attraction. Our findings provide evidence for a negative regulatory role of cFLIP for TNF-dependent apoptosis and partially for epidermal inflammation. However, alternative signaling pathways may contribute to the development of the dramatic skin disease upon cFLIP deletion. Our data warrant future studies of the regulatory mechanism controlling the development of skin disease upon cFLIP deficiency and the role of cFLIP/TNF in a number of inflammatory skin diseases, including toxic epidermal necrolysis (TEN). Tumor necrosis factor (TNF) is a pleiotropic cytokine which is critical for retaining tissue homeostasis by controlling both cell death and inflammation. Several checkpoints decide if TNF will lead to cell death or survival . The firThe skin provides the first mechanical and immunological barrier protecting the body from environmental or microbial danger, and the maintenance of the skin homeostasis is of critical importance . The keyIn this work, we generated and analyzed a double-knockout mouse model lacking TNF completely and cFLIP exclusively in keratinocytes. With this new genetic mouse model, we can confirm that the inflammatory skin disease that results from the epidermal deletion of cFLIP is strongly but not exclusively dependent on TNF. Additional, yet unknown signaling pathways beyond TNF signaling may play a role in the cell death and inflammatory pathogenicity in cFLIP-deficient skin.fl/fl-K14CreERtam mice with TNF−/− mice.We could previously show the role of the TNF autocrine loop for the cell death induced in cFLIP-deficient keratinocytes in vivo . In our fl/fl-K14CreERtam (cFLIP KO), TNF−/−cFLIPfl/fl-K14CreERtam (cFLIP KO/TNF KO), and TNF−/− (TNF KO) animals were epilated, and tamoxifen was applied for 3–5 days on the epilated back skin. Successful deletion of cFLIP fl/fl alleles upon tamoxifen application, as well as TNF deficiency, was confirmed by PCR  demonstrated that TNF deficiency partially protected cFLIP-deficient epidermis from the characteristic acanthosis and spongiosis A. The ty+ T cells at T72H in both cFLIP KO and cFLIP KO/TNF KO animals , known to be expressed in the epidermal layer only during inflammatory disease, demonstrated reduced expression in the epidermis of cFLIP KO/TNF KO mice A. Immuno animals A. Quanti animals B. Intere animals A,C.To gain further insights into the role of TNF for the inflammatory response in epidermal cFLIP KO skin, we next performed gene expression profiling to compare epidermal messenger RNA (mRNA) isolated from TNF KO, cFLIP KO, and cFLIP KO/TNF KO animals at T72H. Focusing on inflammation-related genes involved in the attraction of inflammatory cells, we detected a group of genes known to be related to modulation of T cell activity and attraction ,13, whiccFLIP, FADD, and caspase-8 are key molecules controlling programmed cell death. Our previous studies demonstrated the crucial regulatory function of cFLIP isoforms for the formation and regulation of the intracellular signaling platform called the ripoptosome. This platform is capable of mediating both apoptotic and necroptotic signaling pathways . FADD orIn a previous study, we clarified the role of cFLIP for skin homeostasis and integrity in vivo. We demonstrated that the fulminant cell death in cFLIP-deficient keratinocytes in vivo and in vitro is exclusively apoptotic and can be inhibited by TNF-neutralizing antibodies . Both inOn the basis of the present data and published reports regarding dysregulation of the keratinocyte homeostasis resulting in cell death and inflammation, we postulate the following scenario: NF-κB activation promotes the expression of numerous target genes, including proinflammatory genes, cFLIP (NF-κB inhibitor), and TNF (NF-κB inducer). The LUBAC is critically required to activate NF-κB signaling; therefore, absence of the LUBAC may lead to ripoptosome formation and cell death. cFLIP deficiency results in blocking of NF-κB , promotiPrevious reports using genetic models indicated that dysregulation of cell death and TNF signaling in keratinocytes is a potent inducer of inflammatory skin disease ,19,20,28fl/fl K14CreERtam TNF−/− (cFLIP KO/TNF KO) mice, cFLIPfl/fl K14CreERtam (cFLIP KO) mice [To generate cFLIPKO) mice  were croDNA was isolated from the epidermis using a standard DNA isolation procedure. The presence of the wild-type, floxed, or Cre-excised (deleted) cFLIP allele was confirmed by multiplex PCR. Three pairs of primers were used for simultaneous detection of wild-type (wt), cFLIP floxed (fl), and excised (x) allele as outlined. TNF deletion was confirmed by conventional PCR. Primer sequences are available upon request.Firstly, 72 h after the first tamoxifen application, skin was isolated, formalin-fixed, and paraffin-embedded. The following primary antibodies were used for fluorescence microscopy: K14, K10, loricrin , and cleaved caspase-3 . Secondary biotin-conjugated antibodies were from Vector Laboratories , i.e., Cy-2/Cy-3-conjugated streptavidin. Image processing was applied identically for all samples and controls. Quantification of or immunofluorescence labeling of histological slides was performed semiquantitatively by three independent investigators blinded to the treatment of the respective mouse in at least 20–25 fields of each sample.PKs were isolated from newborn complete skin or adult tail skin and immortalized using spontaneous immortalization CnT-PR medium . Immortalized keratinocytes were transduced with an LV construct expressing Cre-Recombinase or control LV.The pFU Cre SV40 puro W construct was kindly provided by John Silke . Lentivirus-expressing Cre-recombinase was generated in HEK cells.t-test was used for statistical data analysis. Significance is stated whenever p was <0.05.The standard error of mean (SEM) was determined for three independent experiments performed with immortalized keratinocytes pooled from 3–5 newborn animals. Student’s RNA isolation from epidermis or cultured PKs was performed with RNeasy Kit . For complementary DNA (cDNA) synthesis, SuperScript II Reverse Transcriptase  was used with a mixture of random nanomers and oligo dT primers in a ratio 10:1. RT-qPCR analysis was performed using KAPA SYBR Fast qPCR  in the Abi 7300  real-time thermal cycler. Equal cycling conditions were used to amplify genes of interest and reference gene products. Mean values were calculated using data obtained from three independent experiments. Normalization of each experiment was performed to β-actin expression. Primer sequences and details for qPCR cycling conditions are available upon request.mRNA from was isolated from epidermis of 3–6 animals per group and pooled. The Affymetrix GeneChip Mouse Gene 2.0 ST Array  was used according to the manufacturer’s instructions. A Custom CDF V. 22 with ENTREZ-based gene definitions was used to annotate the arrays . Quantil


Antitumour necrosis factor (TNF) therapy has revolutionised treatment of several chronic inflammatory diseases, including spondyloarthritis (SpA). However, TNF inhibitors (TNFi) are not effective in all patients and the biological basis for treatment failure remains unknown. We have analysed induced immune responses to define the mechanism of action of TNF blockers in SpA and to identify immunological correlates of responsiveness to TNFi.Immune responses to microbial and pathway-specific stimuli were analysed in peripheral blood samples from 80 patients with axial SpA before and after TNFi treatment, using highly standardised whole-blood stimulation assays. Cytokines and chemokines were measured in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory, and gene expression was monitored using nCounter assays.2 synthesis and signalling, leucocyte recirculation, macrophage polarisation, dectin and interleukin (IL)-1 signalling, as well as the nuclear factor kappa B (NF-kB) transcription factor family as key pathways targeted by TNF blockers in vivo. Analysis of induced immune responses before treatment initiation revealed that expression of molecules associated with leucocyte adhesion and invasion, chemotaxis and IL-1 signalling are correlated with therapeutic responses to anti-TNF.Anti-TNF therapy induced profound changes in patients’ innate immune responses. TNFi action was selective, and had only minor effects on Th1/Th17 immunity. Modular transcriptional repertoire analysis identified prostaglandin EWe show that TNFi target multiple immune cell pathways that cooperate to resolve inflammation. We propose that immune response profiling provides new insight into the biology of TNF-blocker action in patients and can identify signalling pathways associated with therapeutic responses to biological therapies. Antitumour necrosis factor (TNF) therapy has revolutionised treatment of many chronic inflammatory diseases, including spondyloarthritis and rheumatoid arthritis. However, TNF inhibitors (TNFi) are not effective in 30%–40% of patients. The immunosuppressive effects of TNF blockers therefore expose a substantial fraction of patients to side-effects, in particular infections, without clinical benefit. Despite the extensive use of TNFi for many years, the biological basis for treatment failure remains unknown.We demonstrate that anti-TNF therapy induces profound changes in patients’ innate immune responses, but does not affect Th1/Th17 immunity.2 synthesis and signalling, leucocyte recirculation, macrophage polarisation, dectin and interleukin (IL)-1 signalling, as well as the NF-kB transcription factor family are key pathways targeted by TNF blockers in vivo.Modular transcriptional repertoire analysis showed that prostaglandin ETo investigate the concept that the immune status of patients before treatment initiation will define their response to TNFi treatment, we have searched for immunological transcripts that correlate with clinical efficacy of TNF blockers in stimulated immune cells. We found that high expression of molecules associated with leucocyte adhesion and invasion, chemotaxis and IL-1 signalling is correlated with favourable outcome of anti-TNF therapy.We have established a robust pipeline to monitor immune responses in patients that can be translated into a clinical setting. We show that immune response profiling can identify signalling pathways associated with therapeutic responses to TNFi. Further studies will assess whether this approach can be used to develop molecular biomarkers to help stratify patients to the most appropriate therapy.Chronic inflammatory diseases (CID) are challenging illnesses that often strike at a young age and cause lifelong morbidity, representing a considerable burden for the affected individuals and for society. Spondyloarthritis (SpA) is a family of related inflammatory disorders with common pathological and genetic features.Mycobacterium tuberculosis reactivation.Antitumour necrosis factor (TNF) therapy has proven effective to reduce inflammation and clinical symptoms in SpA; however, little is known about how TNF inhibitors (TNFi) affect immune responses in patients, and TNFi have been associated with infectious complications,Furthermore, the high rate of non-responsiveness (30%–40%) to TNFi exposes a substantial fraction of patients to side effects without clinical benefit, and it is still not possible to determine which patients will respond to TNFi before treatment initiation.Here, we investigated the global impact of TNFi on immune responses to microbial or pathway-specific stimuli, with the goal to enhance our understanding of the molecular mechanism of action of TNF blockers in patients with SpA and to identify immunological correlates of responsiveness to TNFi.Peripheral blood samples were obtained from 80 biologic-naïve patients fulfilling Assessment of SpondyloArthritis international Society (ASAS) criteria for axSpA,Patients’ demographics, HLA-B27 status, information regarding symptoms, ongoing treatments, comorbidities and other main clinical features of SpA were recorded on a Case Record Form before and 3 months (D90) after initiation of anti-TNF therapy see .10.1136/annrheumdis-2020-218304.supp1Supplementary dataPrimary responsiveness to anti-TNF therapy was based on the Ankylosing Spondylitis Disease Activity Score (ASDAS).Whole-Blood TruCulture Stimulation was performed with TruCulture assays .To generate in vitro derived macrophages, monocytes were isolated from healthy donors and cultured with macrophage colony-stimulating factor (M-CSF) in the presence or absence of TNFi. Cells were polarised towards M1 with LPS  and interferon (IFN)-γ , or towards M2 with IL-4 and IL-13 .Quantitative set analysis of gene expression was performed using the R QuSage package.Methods are described in detail in the We analysed immune responses in patients with axSpA with indications for TNFi treatment , using wOnly few secreted proteins increased after TNFi therapy. Among these was IL-10 following stimulation with gardiquimod , a selecThese results show that TNFi induce selective changes in patients’ immune responses, mostly detected in the challenged immune system, and not in the resting state .To determine the early effects of TNFi, we analysed 17 consecutive patients with axSpA 7 days after initiation of TNFi therapy . SecretiThe reduction in proinflammatory mediators was maintained at D90 , demonstTo gain insight into the mechanisms by which TNFi affect immune responses, we analysed the expression of immune-related genes before and at D7 and D90 after TNFi treatment. TNF blockade profoundly altered the transcription of a large number of genes .NFKB1, RELA, NFKB2 and RELB, and NF-kB targets, such as IL1A, IL1B and CCL20 , the key enzyme in prostaglandin E2 (PGE2) biosynthesis and PTGER4 encoding the PGE2 receptor EP4 blockade .Candida albicans and other fungi such as Dectin-2 (encoded by CLEC6A), or Mincle (encoded by CLEC4E) and associated signalling molecules, such as CARD9, a molecule involved in antifungal immunity that mediates signals from CLRs to the NF-κB pathway via BCL10.TNFi therapy also reduced the activity of the ‘dectin’ module , which gWhile gene set activities for most gene modules were reduced by TNFi, we observed increased activity at D7 of the ‘cytotoxic molecules’ module and of the ‘M2-like monocytes’ gene module, while the overall activity of the module ‘M1-like monocytes’ was reduced after TNFi, indicating that TNF blockers may affect monocyte/macrophage polarisation .MRC1, the scavenger receptors MSR1 and CD163, the decoy receptor IL1R2, and of IL10 , and measured transcript levels before and at different time points after stimulation . Several or IL1A .We then asked whether TNFi affect also macrophage gene expression. As the analysis of tissues is rarely performed in axSpA,IL18 .To explore if different types of anti-TNF drugs could have an impact on therapeutic responses to TNFi, we compared differential gene expression between responders and non-responders treated with soluble TNFR2 (n=53) to those treated with monoclonal antibodies (n=27). We found a good correlation (R=0.901) for the 55 genes differentially expressed. These data indicate that the type of TNF blockers does not have a major effect on the genes significantly associated with therapeutic responses before treatment .A search of the DICE databasePLAU and PLAUR, the integrin subunits ITGB1, ITGA5, ITGAX, and ITGA6, and the CD2 ligand CD58 , IL-6 (IL6R) and IL-1  was also substantially higher in responders than in non-responders, as was expression of the IL-1R-associated kinases IRAK1 and IRAK3, and of NLRP3, which controls caspase-1-dependent processing of pro-IL-1β and IL-18. These data indicate that the activation status of the IL-1 signalling pathway may influence responsiveness to TNFi. We also noted substantially higher expression in responders of CLEC5A , an important mediator of autoimmune inflammation in experimental arthritis modelsAmong the 55 differentially expressed genes, 15 regulate key steps of leucocyte migration and invasion: these include and CD58 . The impsponders . ExpressTo investigate immune responses in patients with SpA, we have used highly standardised and robust assays that may be directly translated into a clinical setting. ‘TruCulture’ assays were designed to preserve physiological cellular interactions and capture immune cell activity without introducing sample collection and manipulation variables.Most of the effects of TNFi could be observed only in stimulated cultures, supporting the notion that TNFi act on activated immune cells, rather than in homeostatic conditions. This may explain the relatively modest changes in gene expression in response to TNFi detected in a recent study of unstimulated PBMCs from patients with axSpA.Our modular transcriptional repertoire analysis of the stimulation culturesWe found a strong decrease of proinflammatory molecules produced primarily by innate immune cells, pointing to the importance of these cells in SpA pathogenesis. The decreased activity of the NF-κB module underlines the major role of these factors in mediating TNF-blocker functions. However, TNF blockade had only minor effects on the expression and secretion of IL-6, contrary to what observed in RA patients.IL1R2 and IL1RN, are implicated in the resolution of inflammation and orchestrate tissue repair and remodelling.M. tuberculosis.We observed downregulation of the classical, M1-like module and an increase of the non-classically activated, M2-like monocyte gene module activity, consistent with the finding that TNFi can expand a cell population with a M2 macrophage-like appearance in vivo and in vitro.PTGS2, the key enzyme in prostaglandin E2 (PGE2) biosynthesis and target of non-steroidal anti-inflammatory drugs, the first-line treatment of SpA. PGE2 is an important early mediator of enthesitis, the hallmark of SpA2 induces vasodilation, which may facilitate neutrophil recruitment into the entheseal compartment.2 receptor PTGER4 (EP4) was downregulated by TNFi. Signalling through EP4 upregulates IL-23R expression promoting human Th17 cell development,PTGER4 has been associated with SpA susceptibility, as have been NFKB1 and CARD9,TNFi strongly downregulated expression of 2 biosynthesis via PTGS2 downregulation is of particular relevance for enthesitis, a critical early pathogenic feature of spondyloarthitis, while shifting the balance of macrophages from a proinflammatory phenotype to a proresolving phenotype is important for the resolution of synovitis. MDL-1/CLEC5A was among the most strongly downregulated molecule after TNFi therapy. Dengue virus-mediated activation of MDL-1/CLEC5A can trigger potent induction of TNF, IL-6 and IL-1β and NLRP3 inflammasome activation and shock.Our findings suggest that TNFi target several immune cell pathways that cooperate to control inflammation. Targeting PGEThe involvement of multiple pathways in TNF-blocker functions could also explain the difficulties in identifying a genetic marker for treatment response to TNFi.In conclusion, we suggest that immune response profiling of patients is a powerful approach to define the mechanism of action of biological drugs and may be a useful strategy to establish objective criteria guiding treatment decisions.


The sensory neocortex consists of hierarchically-organized areas reciprocally connected via feedforward and feedback circuits. Feedforward connections shape the receptive field properties of neurons in higher areas within parallel streams specialized in processing specific stimulus attributes. Feedback connections have been implicated in top-down modulations, such as attention, prediction and sensory context. However, their computational role remains unknown, partly because we lack knowledge about rules of feedback connectivity to constrain models of feedback function. For example, it is unknown whether feedback connections maintain stream-specific segregation, or integrate information across parallel streams. Using viral-mediated labeling of feedback connections arising from specific cytochrome-oxidase stripes of macaque visual area V2, here we show that feedback to the primary visual cortex (V1) is organized into parallel streams resembling the reciprocal feedforward pathways. This suggests that functionally-specialized V2 feedback channels modulate V1 responses to specific stimulus attributes, an organizational principle potentially extending to feedback pathways in other sensory systems. In the primate cortex, visual images are processed by multiple hierarchically-organized areas reciprocally connected via feedforward and feedback circuits. Here the authors show that feedback circuits are organized into segregated parallel streams that resemble feedforward pathways. Neuronal receptive fields (RFs) in higher-order areas become tuned to increasingly complex stimulus features, with multiple parallel pathways specialized in processing specific attributes of sensory stimuli . The role of FF connections in shaping cortical RFs has long been recognized2. In contrast, little is known about the function of feedback (FB) connections, although they have been implicated in top-down modulations of neuronal responses, such as attention4, prediction6, and sensory context10. One limitation to understanding the computational function of FB is that we lack information on the rules of FB connectivity to constrain models of FB function. For example, it is debated whether FB connections are anatomically diffuse and, therefore, unspecific with respect to the functional domains they contact in lower-order areas, or whether they are patterned and functionally specific11. A related question is whether diffuse and unspecific FB connections integrate information across parallel processing streams, or whether these connections maintain stream specificity, like their reciprocal FF pathways. Answering these questions would further our understanding of FB function and allow us to refine and inspire theories of cortical computation, many of which make specific assumptions about the functional specificity, or lack thereof, of FB connections16.In the primate sensory neocortex, information travels along multiple parallel feedforward (FF) pathways through a hierarchy of areas18, and CO compartments in V1 and V2 have specialized functional properties and maps20. It is well established that CO blobs project predominantly to V2 CO thin stripes, while V1 interblobs project to V2 thick and pale stripes22. These projections arise predominantly from V1 layers (L) 2/3 and 4B, and sparsely from 4A and 5/6, with L4B projecting more heavily to thick stripes compared to other stripe types24. However, it is debated whether V2-to-V1 FB connections form similar parallel pathways that segregate within V1 CO compartments27, therefore maintaining stream specificity, or diffusely project to all compartments28, thus integrating information across streams. This controversy is primarily due to the lack, in previous anatomical studies, of sensitive anterograde neuroanatomical tracers capable of labeling V2-to-V1 axons fully and selectively, without also labeling the reciprocal V1-to-V2 FF projections. In this work, using selective viral-mediated labeling of FB connections arising from specific V2 CO stripes, we show that, like V1-to-V2 FF pathways, V2 FB connections to V1 are organized into multiple parallel streams that segregate within the CO compartments of V1 and V2. These results suggest functionally specialized FB channels.FB connections from the secondary visual area (V2) to the primary visual cortex (V1) in macaque monkey are well suited to address questions of anatomical and functional specificity and parallel FB pathways. This is because V2 is partitioned into cytochrome-oxidase (CO) stripe compartments that receive segregated FF projections from specific V1 CO compartments and layers8. We quantified the resulting distribution of labeled FB axons across V1 layers and CO compartments. We present results from a total of 13 viral injections made in area V2 of 5 macaque monkeys, spanning all V2 layers. First, we describe the distribution of V2 FB axon terminals across V1 layers (n = 4 injection cases), and subsequently their distribution relative to CO blobs and interblobs (n = 9 injection cases).To selectively label FB connections to V1 arising from specific V2 CO stripes, in 3 animals (7 injections), we first identified the V2 stripes in vivo, using intrinsic signal optical imaging. We then targeted to a particular V2 stripe type injections of a mixture of Cre-expressing and Cre-dependent adeno-associated virus, serotype 9 (AAV9), carrying the gene for green fluorescent protein (GFP) or tdTomato (tdT) (see “Methods”). In 2 animals (6 injections), injections were made blindly with respect to V2 stripe identity, and the stripe location of the injection sites determined postmortem. We have previously shown that in the primate visual cortex, this viral vector combination results in selective anterograde infection of neurons at the injected V2 site and virtually no retrograde infection of neurons in V123, here we term pale stripes located lateral or medial to an adjacent thick stripe as pale-lateral or pale-medial, respectively. Analysis of the laminar distribution of labeled FB axons resulting from these injections was performed on V1 tissue sectioned perpendicular to the layers along an axis parallel to the V1/V2 border. Fluorescent label was first imaged in sample sections, the same sections were then stained for CO, to reveal layers, and finally the sections were re-imaged simultaneously for both CO and fluorescent signals, to maintain perfect alignment of the FB-label and CO-defined cortical layers (see “Methods”). Qualitative observations of sections imaged for GFP and tdT fluorescence  revealed that two AAV9-tdT injections were confined to thin stripes . Within each stripe group, we also statistically compared the resulting FB-label intensity across the sublayers of L4 . We found no significant difference in the amount of FB projections from thin stripes to the different L4 sublayers  showed significantly denser FB terminations compared to L4A (n = 9 bins over 2 injection cases) and 4 C , while L4A did not differ significantly from L4C. In summary, L4B receives significant FB projections from V2 thick/pale stripes, but not from V2 thin stripes, and the latter project less heavily to L6 compared to thick/pale stripes.Layer-by-layer statistical comparisons across stripe groups  revealed a significant difference in the projections to L4B, which were virtually absent after thin stripe injections, but present when the injection encompassed also the pale/thick stripes. Moreover, injections in thin stripes resulted in significantly less projections to L6, compared to injections that additionally encompassed thick/pale stripes , an injection of AAV9-GFP was targeted to a thin V2 stripe, and an injection of AAV9-tdT to the adjacent pale-medial stripe. Figure We next asked how FB projections from different V2 stripe types are distributed across the tangential plane of V1. Figure n = 3), thin (n = 3), or pale (n = 3) stripes.We next asked how the terminal patches formed by FB projections arising from each of the stripe types relate to the CO blobs and interblobs of V1, and whether patchy terminations occur throughout the V1 layers of FB termination. Below we present results from a total of nine viral injections that were confined to V2 thick  that were confined to V2 thick stripes. Analysis of the distribution of labeled FB axons relative to the CO compartments of V1 was performed on tissue sectioned parallel to the pial surface. Fluorescent label was imaged in sample sections through the layers where FB axons terminated, namely L1A, 1B, 2/3, 4B, and 5/6; the same or adjacent sections were then stained for CO to reveal layers and CO blobs, and imaged. Images of each section were vertically aligned in a sequential stack through the depth of V1, by aligning the radial blood vessels  were confined to V2 thin stripes. One example AAV9-GFP injection site and resulting FB label in V1 are shown in Fig. Three viral injections . One example AAV9-tdT injection site confined to a pale-medial stripe is shown in Fig. Three viral injections were confined to V2 pale stripes . Like the individual example cases, the population data shows that FB projections arising from V2 thick and pale stripes terminate preferentially in the interblob regions of V1, across all layers of FB termination . In contrast, the CO-compartment location of FB projections to V1 differed significantly between thick and thin stripes, as well as between pale and thin stripes, and this was the case for all layers of FB termination , except for L4B for the thin versus pale-stripe comparison which did not reach statistical significance (p = 0.053), likely due to the low sample size . Moreover, when all layers data were pooled together for each stripe group (see “Methods”), V2 thick and pale stripes showed no significant difference in the V1 CO-compartment location of their projections (p = 0.801), while thick versus thin, and thin versus pale stripes were significantly different from each other .Figure ion Fig. . MoreoveWe have shown that FB projections arising from the different CO stripes of V2 do not diffusely and unspecifically contact all V1 regions within their termination zones, but rather form patchy axon terminations which largely segregate in the CO compartments of V1: thin stripes project predominantly to blobs, and thick and pale stripes predominantly to interblobs. Moreover, all stripe types send densest projections to layers 1/upper 2 and 5B-6, and sparser projections to lower layer 2 and layer 3. However, only thick and pale, but not thin, stripes project to L4B Fig. .Fig. 8Pa11. Conventional tracers used to label FB axons were either transported anterogradely, but had poor sensitivity and resolution , or had good sensitivity, but were transported bidirectionally , thus labeling the axons of both FF- and FB-projecting neurons. Studies using the first group of tracers injected in areas V2 or MT concluded that FB connections to V1 are diffuse, i.e., they make non-clustered terminations in their target areas, which are non-specific with respect to the CO compartments or functional domains of V130. Curiously, however, one such study28 showed at least one example of patchy FB axon terminations in layer 1 of macaque V1, but the authors did not comment on the modularity of FB connections. Studies using injections of bidirectional tracers into V227 or V311, instead, provided evidence for clustered27 and functionally specific27 FB projections to V1. Due to the bidirectional transport of the tracers used in the latter studies, however, it was unclear whether the axon clustering reflected the termination patterns of FB axons or of the axonal collaterals of the reciprocal FF projection neurons, known to form patchy connections31. For the same reason, it had remained unclear whether FB projections to V1 terminate only in L1 and 6 , or also terminate in layers that send FF projections to higher cortical areas (such as L2/3 and 4B). In a few studies, FB axons were labeled by bulk injections of PHA-L or BDA in V2 or MT, and reconstructed through serial sections34. While these studies were not affected by ambiguity in the interpretation of the origin of axonal label, they, however, provided a limited sample of partially reconstructed FB axons.There has been a long-standing controversy regarding the clustering and specificity, or lack thereof, of FB connections to V1, mainly attributable to the technical limitations of the neuroanatomical tracers used in previous studies35. Specifically, we have used a mixture of AAV9-Cre and Cre-dependent-AAV9 to express fluorescent proteins in V2 FB neurons projecting to V1 without simultaneously labeling the axons of the reciprocal V1-to-V2 FF projection neurons. Our results resolve existing controversies by demonstrating unequivocally that FB connections from V2 to V1 form patchy terminations, in agreement with Angelucci et al.25 and Shmuel et al.27, and that the FB terminal patches are predominantly CO-compartment specific, in agreement with Shmuel et al.27. These results are also consistent with previous demonstrations of patchy and CO-compartment-specific FB connections from area V3 to V1 in macaque11. In particular, we demonstrate that in macaque monkey, FB connections arising from the different CO stripes of V2 form parallel channels that mimic the reciprocal parallel pathways from V1 to V2 in this species21. We also show that, with the exception of L1, the layers that give rise to FF connections to V2  receive reciprocal FB inputs. This suggests that V2 FB axons may make direct contacts with V1 neurons sending FF inputs to V2, a hypothesis that we have recently confirmed using rabies virus-mediated monosynaptic input tracing to label monosynaptic inputs to V2-projecting neurons in V136. However, while L4B has been shown to send at least some sparse inputs to thin and pale-medial stripes37, we only observed significant FB inputs to L4B from thick and pale, but not thin, stripes. It is possible that our method of analysis failed to capture very sparsely labeled FB axons in L4B arising from thin stripes. Moreover, our low sample of injections into pale-medial stripes (n = 1) did not allow us to assess potential quantitative differences in the FB projections to L4B from the two pale-stripe types, as has been previously demonstrated for the L4B to pale-stripe FF projections23. In this study, we have not analyzed L4A for potential FB terminations, as this very thin layer is difficult to study in tangential sections, and in pia-to-white matter sections FB projections to L4A appeared to be, at best, very sparse.To overcome the limitations of previous anatomical studies, in this study we have taken advantage of recent advances in neuroanatomical labeling methods based on the use of viral vectors to deliver genes for fluorescent proteins38.Although here we are proposing the existence of at least three segregated FB pathways from V2 to V1, our study shows that FB projections from thick and pale stripes both terminate in the interblobs, allowing for the possibility that these projections contact the same V1 cells, therefore integrating information across these two parallel streams. However, current studies from our laboratory, using rabies virus-mediated monosynaptic input tracing to label direct inputs to V2-projecting neurons in V1, suggest that FB projections from thick and pale stripes contact predominantly distinct V1 projection neurons50. Our finding of segregated FB projections within these same CO compartments, therefore, suggests that V2 FB connections to V1 do not integrate information across parallel functional streams, and thus across stimulus attributes, but rather modulate V1 responses to the specific stimulus attributes that are processed within their functional stream, an organization that may support, for example, feature-selective attention52, or the feature specificity of extraclassical receptive field effects54.Segregation of V1-to-V2 FF pathways within CO compartments likely reflects specialized function, because CO compartments in V1 and V2 show specialized neuronal responses properties and functional maps. Specifically, the blob-thin-stripe pathway is thought to process surface properties (color and brightness), the interblob-pale-stripe pathway object contours and form, and the interblob-thick stripe pathway object motion and depth56, suggesting this FB organization may exist at multiple levels of the visual pathway. Our results are also consistent with results in mouse visual cortex, demonstrating segregation of functional FB channels from L5 of higher visual areas AL  and PM  to V157, which also matches the functional segregation of the reciprocal FF pathways from V1 to AL and PM58. Modular FB axon terminations have also been demonstrated anatomically in L1 of mouse V1, where FB axons from areas AL and PM form interleaved terminal patches59. The FB patches from AL and PM, in turn align with M2 muscarinic acetylcholine receptor-rich and poor patches, respectively, which correspond to V1 regions specialized for the processing of distinct spatio-temporal features60. Interestingly, M2-positive patches are also found in macaque V1 L1 where they are in register with the CO-poor interblobs60. Together with our results, these studies support the notion of functionally specialized parallel FB pathways.Our finding of segregated V2-to-V1 FB pathways is consistent with previous results of stream-specific organization of corticogeniculate pathways in macaque62. Predictive coding models postulate the existence of different neuronal populations encoding expectations and prediction errors, respectively, at each level of the cortical hierarchy. In these models, inter-areal FB connections carry the predictions, while FF connections carry the error in those predictions13. In terms of the architecture of hierarchical predictive coding schemes, one key attribute is the functional segregation of conditionally independent expectations, so that descending predictions are limited to prediction errors reporting a particular stimulus attribute, but not prediction errors reporting opposite or different attributes. For example, predictions about the orientation of a visual edge should not project to prediction errors units encoding visual motion, because knowing the orientation of an edge does not, statistically, tell us anything about its motion. This suggests that direct descending predictions will be largely specific for stimulus attributes , which is consistent with our finding of FB-specific channels.Our results of segregated FB pathways are consistent with predictive coding theories of FB function. According to these theories, the brain generates an internal model of the world, based on sensory data and prior experience, which is refined by incoming sensory data. The latter is compared to the predicted sensory data, and the prediction error ascends up the cortical hierarchy and refines the higher levels’ modelWhile our results strongly support the existence of functionally specialized parallel FB pathways, it is noteworthy that our method of analysis was designed to extract the densest FB terminations in V1. However, lighter FB axonal label was visible in the regions between the densest labeled terminal patches. Therefore, we cannot exclude that a smaller population of FB axons shows a different terminal pattern, such as projections to all CO compartments of V1 or projections to the opposite channel. This population of FB axons may serve to integrate information across streams, or to carry FB information about the same stimulus feature to neurons in different V1 CO compartments representing that feature, for example orientation in the blobs and interblobs. Similarly, FB from multiple stripes could affect the same neurons within the same stimulus feature, for example both thick and pale stripes have orientation maps, and FB to V1 from these stripes may be combined on the same V1 cells on the basis of orientation preference.AAV9 vectors carrying the gene for either GFP or tdT were injected into specific V2 stripes, which in 3 animals were identified in vivo by intrinsic signal optical imaging. In two animals, instead, injections were made blindly with respect to stripe type. The V1 laminar and CO-compartment distribution of FB axons labeled by these viral injections was analyzed quantitatively.Macaca fasciculari; 4 females, 1 male) were used in this study. All procedures involving animals were approved by the Institutional animal Care and Use Committees of the University of Utah and conformed to the guidelines set forth by the USDA and NIH.Five adult (aged 2–5 yrs old) cynomolgus macaque monkeys . In two animals  small durotomies were made just caudal to the posterior edge of the lunate sulcus, over V2, and viral vectors were injected into V2. In the reminder of cases , optical imaging was used to identify the V2 stripes prior to the injections. In the latter cases, a large craniotomy and durotomy  were performed to expose V2 and parts of V1, a clear sterile silicone artificial dura was placed on the cortex, and the craniotomy was filled with a sterile 3% agar solution and sealed with a glass coverslip glued to the skull with Glutures . On completion of surgery, isoflurane was turned off and anesthesia was maintained with sufentanil citrate . The pupils were dilated with a short-acting topical mydriatic agent (tropicamide), the corneas protected with gas-permeable contact lenses, the eyes refracted, and optical imaging was started. Once the V2 stripes were functionally identified , the glass coverslip, agar, and artificial dura were removed and the viral vectors were injected in specific V2 stripe types, using surface blood vessels as guidance. On completion of the injections, new artificial dura was placed on the cortex, the craniotomy was filled with Gelfoam and sealed with sterile parafilm and dental cement, the skin was sutured and the animal was recovered from anesthesia. Animals survived 3–4 weeks post-injections, and underwent a terminal 3–5 day optical imaging experiment to obtain additional functional maps. At the conclusions of this experiment the animal was sacrificed with Beuthanasia  and perfused transcardially with saline for 2–3 min, followed by 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min. One animal was perfused with 2% paraformaldehyde for 6 min and the brain postfixed overnight.Animals were pre-anesthetized with ketamine , intubated with an endotracheal tube, placed in a stereotaxic apparatus, and artificially ventilated. Anesthesia was maintained with isofluorane (0.5–2.5%) in 100% oxygen, and end-tidal COn = 2), or were located in an unidentifiable stripe (n = 4). The viral vectors consisted of a 1:1 mixture of AAV9.CaMKIIa.4.Cre.SV40 and either AAV9.CAG.Flex.eGFP.WPRE.bGH or AAV9.CAG.Flex.tdTomato.WPRE.bGH , which were mixed and loaded in the same glass micropipette (tip diameter 35–45 µm), and pressure injected using a picospritzer. For the injection cases used to investigate the distribution of FB terminals across V1 layers , we slowly injected (6–15 nl per min) 105–150 nl of the viral mixture at a cortical depth of 1.2 mm from the pial surface. After a 5 min pause, the pipette was retracted to a depth of 0.5 mm, and an additional 105–150 nl of the viral mixture was injected at the same speed. The pipette was left in place for an additional 5–10 min before being retracted. These injection parameters yielded injection sites 1.3–1.8 mm in diameter encompassing all cortical layers. For the cases used to investigate the distribution of FB terminals relative to the V1 CO compartments, we used a similar procedure, but we injected smaller volumes in order to keep the injection site confined to a V2 stripe: 15–30 nl were injected at 1.2 mm depth, and an additional 15–30 nl at 0.5–0.6 mm depth. Resulting injection sites ranged in diameter between 0.56 and 0.95 mm and encompassed all V2 layers. One case used for the CO-compartment analysis  were injected, or injections of the same vector were spaced at least 10 mm apart, to ensure no overlap of the resulting labeled fields in V1. In one case MK356), instead, two injections of the GFP-expressing vector were made in a thick and a thin stripe within the same V2 stripe cycle  under red light illumination (630 nm). To identify the V2 stripes, we imaged V2 during presentation of gratings varying in orientation and SF. Orientation maps were obtained by presenting for 4 s full-field, high contrast (100%), pseudorandomized, achromatic drifting square-wave gratings of eight different orientations at 1.0–2.0 cycles/° SF, moving back and forth at 1.5 or 2 cycles/s in directions perpendicular to the grating orientation. Responses to same orientations were averaged across trials, following baseline correction, and difference images were obtained by subtracting the responses to two orthogonally oriented pairs , the block was cut perpendicularly to the layers and parallel to the V1–V2 border. This sectioning plane allows to easily identify layers, as well as the sequence of V2 stripes, which run perpendicular to the border. For the reminder of the cases (n = 9), which were used for the analysis of FB terminals relative to the V1 CO compartments, instead, the block was postfixed between glass slides for 1–2 h to slightly flatten the cortex in the optically imaged area, cryoprotected, and frozen-sectioned tangentially, parallel to the plane of the imaging camera. Sections were wet-mounted and imaged for fluorescent GFP and tdT-labeled cell bodies in V2 (the injection sites) and FB axons in V1, using either Zeiss Axioskop 2 or Axio Imager Z2 microscopes . After digitizing the label, in most cases, the same sections were reacted for CO to reveal the cortical laminae and V1 and V2 compartments, and then re-imaged under bright field illumination (×1.25–5 magnification). In three cases, instead, the location of labeled axons for 2–3 sections was determined by alignment to immediately adjacent CO-stained sections, rather than from the same section imaged for fluorescent label. In four cases, 1–2 sections were further immunoreacted to enhance the GFP signal strength, and then imaged a third time using both bright field and fluorescent channels to maintain a perfect alignment between the CO and label images. GFP immunohistochemistry was performed by incubating sections overnight at 4 °C in a chicken anti-GFP primary antibody , followed by incubation in Alexa Fluor 488 Donkey Anti-Chicken IgG secondary antibody .Areas V1 and V2 were dissected away from the rest of the visual cortex. The block was postfixed for 1–2 h, sunk in 30% sucrose for cryoprotection, and frozen-sectioned at 40 µm. For the cases used to investigate the distribution of FB terminals across V1 layers  by registering the radial blood vessels sequentially in a stack encompassing all cortical layers . IR-tweak is an interactive, multithreaded, application for manual slice-to-slice registration. As control points are placed by the user in one image, their locations in the other image are estimated by the current thin-plate spline transform parameters65. When the user corrects the locations of estimated points in the second image, the transform parameters are updated. CO staining was used to assign injection sites to specific V2 CO stripes. To determine the CO stripe location of the injection sites, typically 3–6 images of serial CO sections were overlaid by aligning the radial blood vessels and merged in Adobe Photoshop. This approach reveals a more complete stripe pattern than any individual CO-stained section, in which the stripes can be incomplete. V2 injection sites were outlined through multiple images of serial injection sites, spanning the depth of the cortex; a composite injection site encompassing all outlines in individual sections was overlaid onto the merged CO stack, and its size and stripe location were determined. The size of the injections was defined as the region at the injection site where labeled cell bodies could be discerned, as the viral vectors we used infect neuronal somata. Dark CO stripes were classified as thick (or thin) if they appeared thicker (or thinner) than the two adjacent dark stripes in CO staining and coincided with thick (or thin) stripes as defined in the optical imaging maps , for cases in which imaging was available. To align CO-stained tissue and fluorescent images of the injection sites to the optical maps, we warped (using IR-tweak software) the most superficial tangential tissue sections containing the surface vessels  to the image of the cortical surface vasculature obtained under green light . Each subsequent section was then aligned sequentially to the top sections by aligning the radial blood vessels . This alignment procedure is illustrated in Supplementary Fig. Digitized fluorescent label and CO staining from the same or adjacent sections were warped, using n = 4), the V1 laminar distribution of labeled V2 FB axons was analyzed in 3 tissue sections , of which one section was selected near the center of the FB labeled field, where FB terminations are densest, and 1 section was selected on each side of this center section, equally spaced across the full extent of the labeled field in L4B . Sections were imaged for GFP and tdT fluorescent label, stained for CO , then re-imaged for both CO staining and fluorescent label. The layer boundaries were traced manually using CO staining. For each section selected for quantitative analysis, a ROI  was centered on the area that contained the densest fluorescent label. The ROI was further binned from top of the cortex to the white matter border into 100 bins. Individual pixel fluorescent intensity values were measured within each bin, and averaged to obtain the label intensity per bin. The different bin intensities were then normalized to the maximum bin intensity in that ROI. The binning of the ROI provided a FB-label intensity profile across the cortical depth. The normalized label intensity in each bin was finally averaged across the 3 section ROIs and, again, normalized to the max average intensity to produce a single V1 laminar profile of the label intensity for each injection case. Population laminar profiles for FB arising from the same V2 stripe type were obtained by averaging the laminar profiles across cases with injections in the same stripe type. The cortical layer boundaries derived from the average layer thickness across CO-stained sections were then superimposed to the label laminar profile , due to the varying number of bins per layer and the non-normal distribution of the data.To determine whether viral injections in different V2 stripe types produced different laminar profiles, we performed statistical comparisons on a layer-by-layer basis. Because layers can vary in thickness across different sections, and therefore ROIs, in this analysis the same layer could contain slightly different numbers of bins across ROIs, each having an average pixel intensity value. The n for the sublayer dataset consisted of the average intensity value of each bin in that sublayer across all ROIs, pooled across all cases with an injection in the same V2 stripe. For statistical comparison we used the Kruskal–Wallis test with a Bonferroni corrected α value of 0.017.For each stripe group, we also statistically compared the resulting FB-label intensity across the sublayers of L4 . The n = 9 injections, 3 for each stripe type), digitized images of fluorescent label and CO staining were warped and aligned into a sequential stack through the depth of the cortex, as described above for the analysis of V2 injection site location. CO staining was used to identify layers, as well as the blobs and interblobs in the superficial layer sections. For each case, we selected for quantitative analysis at least one section (the one with the densest label) through each of the following layers: 1A, 1B, 2/3, 4B, 5/6. However, for some layers , in some cases it was necessary to select for analysis two adjacent sections, because the cut was not perfectly tangential and therefore the label field in that layer was split between adjacent sections; this ensured that the entire labeled FB field for that layer was analyzed. To quantify the distribution of labeled FB axons across V1 CO compartments within each of the chosen sections, we extracted from a L2/3 CO-stained section in the aligned section stack the CO blob borders, blob centers and interblob center-lines  distance from radial distances measured at 20° increments around the blob center. A grand average Bctr–Ictr distance was then computed from all relevant blobs’ mean Bctr–Ictr distances. This grand average distance was doubled to obtain the ROI for that case. This ROI was then centered on each blob in the CO map, and the CO-staining intensity was measured for each pixel within each blob ROI, summed across all blob ROIs, and normalized to the maximum summed intensity value, to produce a CO-intensity heatmap for the case  intensity for each layer of FB termination, by measuring fluorescent intensity within the same size ROI used to compute the CO heatmap, centered on each blob in the CO map that overlaid the labeled FB field in that layer  to the center of the interblob (1.0 on the x-axis) within a 200 µm-wide window centered in the region of highest signal intensity in the heatmap (black boxes in Supplementary Fig. From the heatmaps, we generated graphs of the normalized CO or fluorescence signal intensity as function of distance from the blob center (Supplementary Fig. To produce population heatmaps for each cortical layer Fig. , the layα value of 0.017. We also performed a statistical comparison by pooling all the selected bin locations across all layers for a given stripe type and comparing across stripe types, again using Kruskal–Wallis test with Bonferroni correction.We statistically compared the location of densest FB label produced by injections in different stripe type. To this purpose, for each case layer heatmap, within the 200 µm window of densest label, out of the 20 bins we selected for analysis those with >70% intensity. The locations of the selected bins relative to the blob center were compared layer by layer across stripe types. For statistical comparison across stripes we used a Kruskal–Wallis test with a Bonferroni corrected Further information on research design is available in the Supplementary FigureReporting Summary