Pyogenic granuloma is one of the inflammatory hyperplasia seen in the oral cavity. The term is a misnomer because it is not related to infection and arises in response to various stimuli such as low-grade local irritation, traumatic injury, or hormonal factors. It is most commonly seen in females in their second decade of life due to vascular effects of hormones. Although excisional surgery is the treatment of choice for it, this paper presents the safest and most minimally invasive procedure for the regression of pyogenic granuloma. Pyogenic granuloma is a hyperactive benign inflammatory lesion that occurs mostly on the mucosa of females with high levels of steroid hormones. It is generally believed that female sex hormones play important roles in its pathogenesis , 2. It i The term “pyogenic granuloma” is a misnomer because the lesion does not contain pus and is not strictly speaking a granuloma , 3, 5, 8A female patient of 28 years old came to our outpatient department with a chief complaint of bleeding gums particularly in the lower left premolar region while brushing and chewing food. She also gave a history of asymptomatic soft tissue growth in the mandibular premolar area which was increasing in size gradually since 4 weeks. Patient's medical history was noncontributory, and on clinical examination there was a smooth exophytic lesion manifested as a small erythematous papule on a pedunculated base which is hemorrhagic with spontaneous bleeding on probing the area with soft tissue growth Figures  and 2. TSo by considering all the above features a provisional diagnosis was made as pyogenic granuloma and incisional biopsy was performed under local anaesthesia and sent for histological examination . Based oThe treatment decided was complete scaling and curettage especially in the premolar area where there was soft tissue growth. There was severe bleeding while doing scaling and curettage. However, bleeding stopped within few minutes by applying pressure with gauze. The patient was advised to perform and maintain thorough oral hygiene by brushing twice a day and to use chlorhexidine mouth rinse of 0.12% twice daily.On observation, there was gradual reduction in the growth after the first week . So, it With the presentation of this paper it can be concluded that the combinations of various etiological factors might have caused the inflammatory tissue to cross the threshold from regular gingivitis to granuloma formation. The lesion was painless as nerves do not proliferate within the reactive hyperplastic tissue. It does not necessarily always require invasive excisional treatment; although surgery is successful in minimizing the recurrence of lesion, it often results in functional and esthetic impairment of the soft tissue morphology. So, the consideration should also be given to simpler and noninvasive treatment protocol procedures that resolve the lesion while preserving and improving the mucogingival complex.


The anthracene emission can be recovered via protonation of the amine as the protonated aminomethylene as an electron-withdrawing group that suppresses the PET process. Similarly, chelation between the ligand and the metal ions can also suppress the PET process and results in a fluorescence enhancement (CHEF). When solvents are introduced as the third control, a molecular 2:1 multiplexer is constructed to report selectively the inputs. Therefore, a molecular 2:1 multiplexer is realized in a simple molecular system. SinceIn our previous work, up to seven binary logic gates were realized within a single molecule, in which redox-active tetrathiafulvalene (TTF) was utilized as a switch to control the fluorescence . Herein,In the present work, the fluorescence of anthracene in the simple molecule 1-[(anthracen-9-yl) methylene] thiosemicarbazide L,  47,48] ,48 47,482.1H- and 13C-NMR (TMS) are recorded on a Bruker Avance II 500MHz spectrometer. The mass spectra were measured on a Waters Quattro micro TM API mass spectrometer. Elemental analyses were performed on a Vario EI Elementar system.The UV-vis absorption spectra were recorded on a Shimadzu 2500 UV-VIS spectrophotometer. The fluorescence spectra were recorded on a Shimadzu RF-5301 spectrofluorophotometer using 5 nm input and 5 nm output width. 3.3.1.1H-NMR (DMSO-d6) δ = 7.58 , 7.65 , 7.73 , 8.15 , 8.32 , 8.58 , 8.71 , 9.34 , 11.66 . 13C-NMR (DMSO- d6) δ = 125.2, 125.5, 126.0, 127.8, 129.4, 129.9, 130.1, 131.4, 142.7, 178.6. IR (KBr): ν = 3436 (NH2), 3250 (NH), 1601 (C=N), 1285 (C=S) cm−1. ESI-MS, m/z (%): 279 [M+]. EA (%): C16H13N3S, calcd.: N, 15.05; C, 68.82; H, 4.66; found: N, 15.25; C, 68.61; H, 4.80.When anthracene-10-carbaldehyde was treated with thiosemicarbazide in methanol, the ligand L was readily formed as an orange microcrystalline solid appearing in the reaction medium, and it was characterized with elemental analysis (EA), IR, MS, and NMR spectra data. 3.2.The photophysical properties of the ligand L were initially examined in tetrahydrofuran (THF) and methanol. As shown in PET = −0.1 eV) ) and 659.2 ([CuL2(H2O)2]). To understand the chelation ability of the ligand towards Cu2+, the HOMO (high occupied molecular orbit) and LUMO (lowest unoccupied molecular orbit) distribution of the L were determined by density functional theory (DFT) calculations. As shown in The emission enhancement is significant under the addition of acid to the THF solution of the ligand, while the emission is slightly enhanced in the methanol solution. The enhanced fluorescence upon protonation at amine group is caused by the sufficient protons which disturb and suppress the PET process and result in a recovered emission ,32,33. T2+ in high dielectric and polar methanol. The communication between paramagnetic Cu2+ and fluorophore is predominant, and thus the fluorescence is quenched.In methanol solution, however, the fluorescence is quenched. This might be due to the intrinsic fluorescence quenching by mechanisms inherent to paramagnetic species Cu3.3.Fluorescence is one of the most widely employed signals owing to its high sensitivity, feasibility in detection, and low cost in operation. Since systems containing fluorophores can be switched between emissive state and quenched state via manipulation of the PET process, the chemical system described above could be a simple functional model of logic gate with tuning the fluorescence as output signal.It is interesting to note that the logic analysis of the present system is complex if the solvent is considered as the control input. In this context, the logic system described here is a chemical model of a 2:1 multiplexer. Physically, a multiplexer is a communication device that combines multiple inputs into an aggregate signal to be transported via a single transmission channel. Simply, it is a data selector which outputs any one of several possible inputs via a control switch.2+ while solvent as the control, changes of the fluorescent emission of the ligand corresponding to two different inputs mimic the digital selection function (2+, however, the intensity is still below the threshold and thus it is denoted as output 0. When both proton and Cu2+ are added into the system simultaneously, the fluorescent emission is remarkably enhanced, due to the coordination of the metal ions with the protonated ligands. It means in THF the ligand responses to the input H+.In the present case, the solvent as the third input acts like a mechanical rotary switch to control the logic functions of the molecule, enabling the molecule to output selectively the desirable data from all the inputs. With the input of the proton and the metal ion Cufunction . In THF 2+ quenches the emission of the ligand L. Although the fluorescence is enhanced with the proton added, it is quenched and the intensity is below the threshold when the protons and Cu2+ are both introduced, indicating that the system is selectively responsive to Cu2+ even in the presence of the proton. For the truth table calculation, the emission results in MeOH could be interpreted by using the negative logic rule. Combined with the fluorescent response in both MeOH and THF solvents, the chemical system is capable of mimicking a 2:1 multiplexer function with the solvent switch (In digital design of a multiplexer, where both the inputs and outputs are voltages, the switch for the control is usually an analogous of the mechanical rotator. However, in most molecular logic gates and devices, chemical inputs and photonic outputs are applied, and it is reasonable for the chemical model of molecular devices to choose the solvents used as control switch. If the use of solvent THF is set as the Off state, corresponding to the Off state of a mechanical switch, and the use of MeOH solvent is corresponding to the switch On state, the resulting output of the present system is in agreement with the selective response to any functions of the input sets. In MeOH solution, Cut switch , Table 24.In summary, we have demonstrated a combinational logic circuit of molecular multiplexer within a simple molecular system, where the emission of anthracene and the solvent effect are employed as signaling and switch control, respectively. The work presented herein opens the possibility for further development of chemical logic systems, and thus for the construction of molecular digital devices which are likely to be evolved into components of future molecular computers such as a wet computer. Further investigation of this topic is underway in our lab.


Mycobacterium tuberculosis (M.tb) represents a leading global health concern, with 8.7 million newly emerging cases, and 1.4 million reported deaths annually. Despite an estimated one third of the world’s population being infected, relatively few infected individuals ever develop active clinical disease. The ability of the host to remain latently infected while preventing disease is thought to be due to the generation of a robust type 1 immune response in the lung, capable of controlling, but not clearing, M.tb. A key feature of the type 1 immune response to M.tb is the formation of immune cellular aggregates termed granuloma. The granuloma structure has long been considered a hallmark of host’s protective response toward M.tb. Historically, a correlative relationship between granuloma formation/maintenance and bacterial control has been seen in models where disrupted granuloma formation or structure was found to be fatal. Despite this established relationship much about the granuloma’s role in M.tb immunity remains unknown. Recent publications suggest that the granuloma actually aids the persistence of M.tb and that the development of a necrotic granuloma is essential to person-to-person transmission. Our group and others have recently demonstrated that enclosed within the granuloma is a population of immunologically altered antigen-presenting cells and T lymphocyte populations. Of note, the ability of these populations to produce type 1 cytokines such as interferon-gamma, and bactericidal products including nitric oxide, are significantly reduced, while remaining competent to produce high levels immunosuppressive interleukin-10. These observations indicate that although the chronic granuloma represents a highly unique environment, it is more similar to that of a tumor than an active site of bacterial control. In this review we will explore what is known about this unique environment and its contribution to the persistence of M.tb.Pulmonary tuberculosis, caused by  Mycobacterium tuberculosis (M.tb) is the leading cause of death due to a bacterial pathogen and is responsible for 1.4 million deaths annually, latently infecting one third of the world’s population  caused by infection with pulation . Despiteon .The formation of granuloma is a dynamic process that begins shortly after infection and continuously evolves over time. Temporally, the granuloma can be divided into three distinct phases: (1) the “innate granuloma,” a loose aggregate composed primarily of recruited macrophages and neutrophils; (2) the “immune granuloma” formed following the emergence of antigen-specific T cells; and (3) the “chronic granuloma,” resulting from distinct morphological changes in granuloma structure  initiating the early inflammatory response. While amplifying the host immune response, the recruitment of innate immune cells inadvertently provides a large number of new targets for M.tb to infect and is thought to contribute to the early dissemination of M.tb , interleukin (IL)-6, and IL-8 which facilitate the recruitment of new macrophages and granulocytes to the site of infection and lead to the formation of the “innate granuloma”  led to the activation of the epithelium, which facilitated the recruitment of macrophages to the site of infection through inducing the production of matrix metalloproteinase-9 (MMP-9)  . In murioma size . These fthe host . This vithe host .M.tb infection, the majority of bacilli and antigen reside within an endosome, and are most efficiently loaded onto major histocompatibility complex (MHC) class II . Within the MLN antigen-loaded APCs activate antigen-specific T cells. Due to the nature of class II . The loaresponse . AlthougM.tb infection is highly similar between the mouse and man, the structural formation of the “immune granuloma” differs significantly. In mice, many of the hallmark features of the human granuloma are missing, and thus the knowledge from murine granuloma research should be interpreted with caution. Despite these limitations, animal models have provided significant insight, and have been invaluable in delineating the stages of granuloma formation.The continuous production of chemokines by infected lung APCs efficiently recruits newly primed T cells into the lung. Once in the lung, recruited T cells surround and wall-off infected macrophages, activate them for enhanced bactericidal function, and physically limit their mobility to restrain bacterial dissemination. Indeed, the arrival of effector T cells and the establishment of the classical “immune granuloma” is associated with a plateau in bacterial growth . While tM.tb persistence than its elimination. Despite the fact that animal models do not accurately replicate human granuloma structures, several murine studies have provided invaluable insight into the role of granuloma in preventing M.tb dissemination control. Early studies demonstrating the role of critical cytokines such as TNF and IFN-γ perpetuated the notion that the granuloma was essential for bacterial segregation and limiting bacterial growth in the lung as the absence of either cytokine led to ill-formed granuloma and increased bacterial infection , there is also a failure of granuloma formation, and despite this defect, mice are protected for the first 90 days post-infection, with no increase in bacterial growth compared to wildtype mice within this time frame . This eventual necrosis of the granuloma is now accepted as a necessary event in facilitating the transmission of M.tb by disrupting the lung structure and allowing M.tb to gain access to the major airways. It should be noted that, in addition to person-to-person transmission, the active granuloma leaking bacteria into the airways may also allow for intrapulmonary dissemination  has potent cytotoxic effects and has been implicated in the generation of central necrosis of the granuloma and the transmission of M.tb (M.tb during the development of central necrosis and cavitation (M.tb to the extent of necrosis and cavity formation (M.tb facilitates the necessary structural changes to ensure its own transmission, which occurs at a time when the immune system is most vulnerable.While it is well-known that changes to the granuloma structure are required for bacterial transmission, it is currently unknown whether the host or bacteria are responsible for these changes. Recently, studies have examined the granuloma at early and chronic stages of disease revealing a dramatic shift in the genes expressed by both c stages . Coincidlearance . This issistence . While tironment . This satabolism . Notably of M.tb . Recentl of M.tb . The eng of M.tb . Interesvitation . Indeed,ormation . Moreoveormation . Given tM.tb efficiently hijacks the granuloma to provoke the generation of an immunologically sheltered niche to reside within and persist until the situation is favorable to bacterial transmission.While the true nature of the granuloma still remains to be defined, it is now clearly evident that the granuloma is not just a host-mediated entity of segregation and rather, it is a dynamic battlefield bearing the scars left both by the pathogen and the host immune response. While it may have been originally destined to restrain bacterial dissemination, 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.


Leishmania donovani-induced hepatic granulomas, as well as their importance in protecting against inflammation-associated tissue damage and the generation of immunity to rechallenge. Finally, we discuss the role that computational, agent-based models may play in answering outstanding questions within the field.In human and canine visceral leishmaniasis and in various experimental models of this disease, host resistance is strongly linked to efficient granuloma development. However, it is unknown exactly how the granuloma microenvironment executes an effective antileishmanial response. Recent studies, including using advanced imaging techniques, have improved our understanding of granuloma biology at the cellular level, highlighting heterogeneity in granuloma development and function, and hinting at complex cellular, temporal, and spatial dynamics. In this mini-review, we discuss the factors involved in the formation and function of  Leishmania donovani and L. infantum. These parasites establish long-term infection within multiple organs including the spleen, liver, and bone marrow. In humans and dogs, VL is invariably fatal if untreated, but subclinical infections are common and are associated with granuloma formation , a parasitic disease impacting on health and economy in developing countries, is caused by ormation . In expeormation . TogetheFigure 1. Fundamental insights into the role of different immune cells, effector and regulatory cytokines, and other mediators have been made through the use of gene-targeted mice  are at the heart of the hepatic granuloma, often fusing with each other as a result of migration from neighboring sinusoids . The siggfp/+ mice  play a significant amplifying role. iNKTs-deficient mice have impaired granuloma maturation, lower inflammatory cytokine expression, and reduced expression of CCL2, CXCL5, and CXCL2 . iNKTs ip/+ mice  could beL. donovani-induced granuloma and granuloma maturation . Both CD4+ and CD8+ T cells are indispensable in the effective formation and function of granulomas and for parasite clearance (L. major infection the effector function of a few antigen-specific CD4+ T cells is enhanced by bystander activation (+ T cells within infected livers display an activated phenotype (CD44hi), with ~30–40% of CD4+ T cells having the capacity to produce interferon-gamma . These data are consistent with a model whereby local bystander activation operates within (and possibly even between) granulomas to enhance effector function. Future studies should aim to test such a model and determine whether non-specific T cell recruitment is beneficial for the outcome of Leishmania infection. Furthermore, the extent to which T cell functional differentiation occurs within the granuloma environment remains open.T cells are the predominant cell type present within the learance . 4D intrlearance . Antigentivation . We haveL. donovani-induced granulomas but do not appear to be infected. In contrast, both infected and uninfected monocytes are recruited to BCG-induced hepatic granulomas  antibody resulted in impairment of early antileishmanial resistance followed by delayed granuloma maturation  are also found within L. donovani-induced granulomas although they were shown not to be the targets of effector CD8+ T cells (+ cells in chronic granulomas displayed decreased expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules whereas CD11c+ cells within acute granulomas could support the reactivation of recruited antigen-specific CD4+ T cells and could induce IFN-γ responses from naïve T cells  deficiency where high liver parasite burdens do not result in liver pathology until the severely delayed and exaggerated inflammatory response begins, resulting in hepatic necrosis . Howevernfection . Of inteturation . FurtherL. donovani-induced granulomas and displayed immunoregulatory properties  cells have been shown to be present within operties . HoweverL. donovani-induced granulomas and/or protect against pathology is unknown and is an important question to address. We have shown ~2–3% of CD4+ T cells within infected livers co-express IFN-γ and IL-10, indicative of a regulatory phenotype (manuscript submitted). Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is expressed by Tregs and is important for executing their immunosuppressive function  infection (Whether regulatory T cells (Tregs) are recruited to function . We havefunction , one expring EVL , providianulomas . Other snfection  and the nfection . UnderstL. donovani-induced granulomas may also function to control the resolution stage of hepatic granuloma formation. As describe in Figure 1, granuloma maturation peaks around 4 weeks post-infection (p.i.) followed by parasite clearance and the resolution of granulomas, enabling the liver architecture to return to its pre-infection state. As the hepatic granuloma response continues to increase in the first 4 weeks of infection, it would be reasonable to posit that there must be a mechanism(s) in place to act as “a brake” on the immune response and begin the resolution stage of EVL. Whether the various cell types mentioned in the previous section play a role in this is an interesting topic that has not been previously addressed.Regulatory cells within + T cells and resulted in accelerated granuloma development at infected foci . The study of granuloma formation is invariably linked to both the structure of the granuloma, and the spatial environment in which the granuloma is present. ABM allows for spatial representation of agents , in addition to functional abilities/states for each agent , providing the potential to compare biophysical and functional mechanisms. These models can represent agents individually with their own state providing heterogeneity within a population of agents, allowing for the study of small differences in individual agent behavior that manifest at the population level. Individual agent representation also provides the ability to interrogate and track agents. For example, this facilitates the study of cell recruitment into a granuloma, with respect to the surrounding environment, and the analysis of various interactions and environmental cues received by those cells, resulting in a powerful predictive tool . The choice of ABM approaches should always match the purpose for the model. For example, modeling large populations of homogenous cells or molecules to study population level properties may lend itself more, in terms of efficiency, to mathematical approaches, although frameworks to efficiently model large homogenous populations with ABMs do exist . This ABM provided a means for evaluating whether granuloma heterogeneity might reflect competition for NKT cells or KC diversity (L. donovani infection (manuscript submitted). This model suggests individual granulomas have a heterogeneous capacity for parasite clearance, and predicts that KC autocrine IL-10 is a key regulator of intra-granuloma effector mechanisms. Recently, we have developed a tissue-scale ABM to investigate the dynamics of NKT cell recruitment and KC stimulation during early infection, providing insight into interventions for the promotion of an early granulomatous response. Our model uses artificial sinusoidal environments, generated using published imaging and statistical analysis data (Our initial iversity , but wassis data  and demoLeishmania donovani-induced granulomas represent an intriguing example of innate and adaptive immunity combining in a unique microenvironment to eradicate an intracellular pathogen. Each granuloma represents a highly organized structure and studies discussed in this review have shown that several different cell types and factors function within this complex structure to deliver effective parasite clearance, without causing excessive tissue damage. Interestingly, it is still unknown if granuloma formation is indispensable for immune protection but granulomatous inflammation likely provides the most efficient mechanism of delivering key antileishmanial processes in a focused manner. The advancement of imaging techniques has allowed us to study granulomas in greater detail, highlighting the dynamic and complex nature of these microenvironments, but as a result have raised important questions. The ability of ABMs to create an integrated system, incorporating the multiple factors involved in granuloma formation, offers a powerful tool to help address these questions, enabling the testing of hypotheses and discovery of potential interventions. Future studies should aim to use a combination of experimentation and modeling to unearth the complexities of the Leishmania granuloma and discover how exactly this response delivers its effective antileishmanial function.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.


More than 40 years have passed since Kawasaki syndrome (KS) was first described. Yet KS still remains an enigmatic illness which damages the coronary arteries in a quarter of untreated patients and is the most common cause of childhood-acquired heart disease in developed countries. Many gaps exist in our knowledge of the etiology and pathogenesis of KS, making improvements in therapy difficult. In addition, many KS features and issues still demand further efforts to achieve a much better understanding of the disease. Some of these problem areas include coronary artery injuries in children not fulfilling the classic diagnostic criteria, genetic predisposition to KS, unpredictable ineffectiveness of current therapy in some cases, vascular dysfunction in patients not showing echocardiographic evidence of coronary artery abnormalities in the acute phase of KS, and risk of potential premature atherosclerosis. Also, the lack of specific laboratory tests for early identification of the atypical and incomplete cases, especially in infants, is one of the main obstacles to beginning treatment early and thereby decreasing the incidence of cardiovascular involvement. Transthoracic echocardiography remains the gold-standard for evaluation of coronary arteries in the acute phase and follow-up. In KS patients with severe vascular complications, more costly and potentially invasive investigations such as coronary CT angiography and MRI may be necessary. As children with KS with or without heart involvement become adolescents and adults, the recognition and treatment of the potential long term sequelae become crucial, requiring that rheumatologists, infectious disease specialists, and cardiologists cooperate to develop specific guidelines for a proper evaluation and management of these patients. More education is needed for physicians and other professionals about how to recognize the long-term impact of systemic problems related to KS. Kawasaki syndrome KS, OMIM 611775) is an acute necrotizing vasculitis of the medium and small-sized vessels, predominantly occurring in children aged 6 months-5 years, with a male-to-female ratio of 1.5-1.8 to 1 and a life-threatening predisposition to involve coronary arteries . KS is t11775 is Although reported all over the world, KS is most highly expressed among Asian populations, mainly of Japanese and Korean descent. In Japan, the 20th nationwide survey documented an annual incidence of 216.9 per 100,000 children aged 0-4 years. This incidence is remarkably greater than in Europe  or in the Caucasian population of the United States of America , supporting the speculation that both genetic predisposition and environmental factors are critical for KS pathogenesis ,12. A hoThough many etiological agents have been proposed, the exact cause of KS continues to be elusive. The rarity of the illness in the first months of life suggests a passive protection by maternal antibodies. Seasonality of cases, nationwide epidemics, and the self limited nature of KS favor an infectious trigger. Although the subject is still much debated, current research appears to indicate that the primum movens of KS might be an infectious agent with superantigenic activity, leading to a massive stimulation of the immune system and to the development of KS in a small subset of genetically predisposed individuals . The disLactobacillus casei cell wall extract-induced coronary arteritis, an animal model of KS [The immune response in KS encompasses both aspects of innate and adaptive immunity with a significant overproduction of different cytokines and activation of endothelial cells. Activation of both B and T cells has been detected together with increased proinflammatory cytokine production, including tumour necrosis factor (TNF)-α, interleukin-1 and interleukin-6 ,17. The el of KS . Many stel of KS ,20. Receel of KS . Geneticel of KS .Genome-wide association studies have been performed to identify novel loci modulating the susceptibility to KS and plausible candidate gene associations have been recognized, including apoptosis regulatory genes, genes implicated in the synthesis of transcription factors and interleukin inhibitors . A recenDiagnosing and treating both "atypical" and "incomplete" forms of KS remain major dilemmas for clinicians evaluating children, especially infants, with prolonged or refractory high fever. Even after 5 years of age, the diagnosis of KS can be very hard to establish. According to the American Heart Association and the American Academy of Pediatrics, "atypical" KS is defined as a child with high fever and symptoms and signs heralding the onset of the disease that are not included in the clinical major criteria of KS, such as acute abdominal pain, pulmonary signs, or gastrointestinal signs . The morConversely, the term "incomplete" KS refers to patients who lack the clinical findings of the disease to fulfill the classic criteria, but present fever, at least two of the typical clinical manifestations and CAA revealed by echocardiography . It is cThe primary goal for pediatricians should be early recognition and treatment of both atypical and incomplete cases before coronary arteries are damaged. Among all KS patients, atypical and incomplete cases have the highest risk of developing CAA if left untreated with IVIG ,28. In cAnother suggestive symptom in recognizing KS is the extreme irritability of the majority of pediatric patients, which is mainly related to aseptic meningitis and is uncommon in other febrile diseases . An inteSince no criteria have been developed that can reliably identify children at higher risk for severe disease at the time of initial presentation, all children diagnosed with KS must be treated at the time of diagnosis. Since both KS etiology and pathogenesis are not completely clarified, the general aims of treatment are to rapidly diminish the inflammation within the vascular system and specifically in the coronary arteries, minimize the incidence and progression of CAA, and prevent arterial thrombosis by inhibiting platelet aggregation . The curIVIG and aspirin therapy should be instituted as soon as possible when KS is diagnosed. If CAA are detected in a child prior to the child's fulfilling all KS diagnostic criteria, treatment with IVIG and aspirin should also be initiated then. When the KS diagnosis is delayed but inflammatory parameters are still high, it is however recommended to treat patients aggressively with IVIG and aspirin, especially young infants, even though the efficacy of this early treatment in preventing coronary artery injuries is uncertain . ApproxiAlthough corticosteroids are the treatment of choice in most vasculitides of adulthood, their role as a first-choice treatment for KS is controversial. A multicenter prospective randomized trial of corticosteroids in primary KS showed that the combination of IVIG and corticosteroids improves clinical course and coronary artery outcome, without causing side effects . HoweverNo specific guidelines are available for the management of refractory KS patients in whom inflammatory parameters do not subside and fever persists or recurs 24 to 48 hours after IVIG infusion, with the possible increased risk of coronary damage. Most of these patients will respond to a second infusion of IVIG , but up to one third of non-responders remain febrile with a persistently high serum CRP level ,48. BecaInfliximab, a chimeric mouse-human monoclonal antibody targeting soluble and membrane-bound TNF-alpha, has been successfully used in patients refractory to IVIG and corticosteroids, even with severe coronary artery involvement, without substantial side effects ,53. NoneThe major sequelae of KS, such as CAA leading to potential occlusion and cardiac ischemia, are dramatically decreased as a significant result of therapy with IVIG. Coronary artery damage is regarded as the hallmark of KS and is the single risk factor that contributes the most to an increase in the mortality risk of this disease in any patient . Other csaccular, if axial and lateral diameters are nearly equal, or fusiform, if symmetric dilation with gradual proximal and distal tapering is seen. When a coronary artery is larger than normal without a segmental aneurysm, it is considered "ectatic". In addition, according to the last American Heart Association statement, aneurysms can be classified as "small" , "medium"  or "giant"  [Aneurysms are usually classified as iameter) .Abnormal endothelial function might persist for many years after the resolution of the acute phase of KS, even in patients without detectable coronary artery damage. Patients with this abnormal endothelial function might display subclinical vasculitis many years after KS . EndotheThe long-term outcome of adults who have recovered from KS without CAA is considered excellent, though longitudinal studies are required before confirming this speculation. A heart-healthy lifestyle is recommended for all patients with a history of KS, including avoidance of smoking, low fat diet, control of blood pressure and lipid panels, and regular physical activity, unless contraindicated ,69. LongRoutine vaccinations are recommended for all individuals with KS. The schedule for administration of inactivated childhood vaccines should not be interrupted. As the passively transmitted antibodies in IVIG can block the specific immune response, it may be necessary to reschedule live viral vaccines  to at least 9 months after IVIG administration. In Japan an interval of 6 to 11 months is recommended between IVIG infusion and live vaccine administration, but there is no final agreement for IVIG-non responders who received a second or a third IVIG infusion; if the child's risk of exposure to measles or varicella is high, the child should be immunized and then reimmunized at least 11 months after administration of IVIG . The admAlthough our understanding of KS has increased enormously and many facts about KS have been elucidated, numerous dilemmas remain unsolved. The first concern is the need for the discovery of the causative agent that may allow more appropriate treatment for affected children. Secondly, there is urgency to identify specific laboratory tests to shorten the gap between the appearance of the first clinical manifestations and the diagnosis, with the possibility of reducing the occurrence of coronary damage or cardiac complications in adulthood. A critical point is related to infants younger than 3-6 months who frequently display an atypical or incomplete form of the disease, often sharing signs and symptoms with other infantile febrile illnesses mimicking KS. Therefore, it is crucial to perform early transthoracic echocardiography in all patients with suspected or established KS, and introduce the appropriate therapy even in the absence of all typical findings.Patients with giant aneurysms require aggressive anticoagulation with lifelong follow-up, including stress tests and multi-slice computed tomography or magnetic resonance imaging. The degree to which KS is a cardiovascular risk factor for progression to atherosclerosis is under debate and long term evolution and consequences of the disease are still unknown, despite many efforts of researchers worldwide. The available data suggest that all KS patients, including those without CAA, should remain connected to their medical system, even in their transition to adult medical care, with the hope that collaborative scientific research among various centers will help define the cause and eventual prevention of the syndrome and the optimal management of current KS patients.KS: Kawasaki syndrome; CAA: coronary artery aneurysms; IVIG: intravenous immunoglobulins; TNF: tumour necrosis factor; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein.The authors declare that they have no competing interests.FF (submitting author) primarily created and drafted the manuscript and revised it based on co-authors' suggestions; SC and DR have been involved in the critical revision of the manuscript, giving the final approval of this final version.


Persons with KS and uncontrolled HIV infection have HHV-8 DNA detected more frequently at mucosal sites and plasma , but it Participants collected daily oral swabs and weekly plasma samples over 4 weeks to quantify HHV-8 DNA by polymerase chain reaction.297 participants collected a total of 8,045 oral swabs and 1,392 plasma samples. HHV-8 DNA was detected in 1,561 (19%) oral swabs and 419 (30%) plasma samples. The frequency of detecting any HHV-8 differed by KS status. HHV-8 was detected in the oropharynx of 70% (64/92) persons with KS vs. 27% (52/194) without KS (p<0.001), and in the plasma of 96% (88/92) persons with KS vs. 20% (38/194) without KS (p<0.001). The median amount of HHV-8 DNA detected in oral swabs was significantly lower in persons with KS (3.2 log copies/ml) than those without KS  (Figure Increased quantities of HHV-8 DNA were detected in the oropharynx of persons without KS and those with poor dentition. The latter observation may be explained if higher CD4 counts allow for increased inflammation in the oropharynx, in turn leading to greater HHV-8 replication. Quantities of HHV-8 are higher in the plasma of persons with either HIV infection or KS, perhaps representing the propensity of HHV-8 to disseminate systemically in the absence of effective immune control or from foci of replication in KS tumors.


DNA barcoding and molecular or integrative taxonomy projects are among the most valuable sources for biobank specimens of wild organisms, thanks to – among other aspects – the high level of specimen diversity and thanks to a thorough taxonomic coverage. Specimens used to build barcoding reference libraries tend to be accompanied by deeper and higher-quality data than samples from many other sources, as they are often contributed by taxonomists, and identifications are cross-checked through barcode analysis. Vouchering of morphological specimens in natural history collections is a prerequisite for proper barcoding, which is advantageous for biobanking as well, as biobank samples should always be linked to specimen vouchers. As a further added value, barcoding provides an inherent, molecular species ID tag to the processed biobank sample.Banked barcoding samples can greatly catalyze taxonomy, as well as many other fields of application, such as the emerging large genome sequencing projects that are constantly increasing the demand for well-preserved samples from a multitude of different species see .Considered from the opposite perspective of the synergy, barcoding can benefit greatly from biobanking as well. Biobanking enables the expansion of barcoding datasets with biobanked samples from other projects. It also offers the possibility to add new barcoding markers any time in the future, e.g. scaling up to ‘next-generation barcoding’ e.g.  if feasiFinally and most importantly, biobanks offer barcoding projects the possibility to adequately voucher their molecular samples and to warrant reproducibility of results.http://www.dnabank-network.org/) or soon also the Global Genome Biodiversity Network (http://ggbn.org/). Those museums and natural history collections that implement these features and commit themselves to provide the community with proper biobanks  offer a very efficient and elegant way to both draw on and to deposit morphological-molecular ‘tandem’ samples. Often underappreciated by public and policy-makers  collected through the vouchers: repetitively or in an additive manner. Put short, vouchers link individual studies with other studies and inferences, past or future.It becomes obvious that it is only through adequate vouchering that we can make organismic biology meaningful, warranting reproducibility and embedding our research into existing and emerging knowledge.In a laudable approach to increasing semantic accuracy regarding the voucher concept, PageBreakhologenophore is the specimen voucher from which the molecular sample is directly derived, an isogenophore is a different specimen with a clonal relationship to the study organism, while a progenophore represents a voucher that is linked to the specimen sampled for molecular analysis by a parent-descendant or sibling relationship. A paragenophore is a putatively conspecific specimen voucher collected together with the ‘molecular’ specimen. The same applies to the syngenophore, except that it is collected at another place or time.a These genealogy-based distinctions made by specimen voucher: a specimen serving as the basis for taxonomic identification and possibly also for other queries. A specimen voucher is often, but not necessarily a whole organism, or part of it .Narrower terms: morphological voucher, acoustic voucher, e-voucher, etc.morphological voucher: a specimen that allows the inspection of morphological characters.e-voucher: digital objects that serve as vouchers , e.g. sound recordings, audiovisual material, images, etc.molecular voucher: a sample that is deliberately preserved and curated in a way that will conserve its molecular properties for analysis. A molecular voucher should always be linked to a specimen voucher .Narrower terms: biobank voucher, DNA voucher, tissue voucher, RNA voucher, protein voucher, genomic sample, etc.tissue voucher: tissue subsampled from a specimen – or the entire specimen –, preserved (usu. frozen) to keep its molecular properties (either fixed tissue or viable cells) for future analysisDNA voucher: the isolated and preserved, frozen or dried (usu. genomic) DNA. As a derived sample, a DNA voucher should not – if anyhow possible – function as specimen voucher.biobank voucher: any molecular voucher curated in a biobank. A biobank voucher is a biobank sample that links to other physical objects or data (other than their metadata), i.e. most biobank samples are (biobank) vouchers, as they usually link to a separate specimen vouchergenomic sample: preserved sample containing (isolated or as a constituent) a high percentage of an organism’s genome in widely unfragmented formPageBreakbiobank: a curated collection/repository of biological materials that warrants long-term integrity at molecular level, authenticity, availability and rights management of its samples by adhering to standard operating procedures (SOPs).Narrower terms: DNA bank, tissue bank, biodiversity biobank, etc.biodiversity biobank: term currently used to refer to a biobank holding non-human samplesgenomic collection: a molecular collection holding genomic samples


Immune reconstitution inflammatory syndrome during anti-retroviral treatment of acquired immunodeficiency syndrome (AIDS) -associated gastrointestinal Kaposi's sarcoma has rarely been reported.A 36-year-old Asian Indian male, newly diagnosed with AIDS and treatment naïve, was started on highly active antiretroviral therapy (HAART). He developed acute intestinal obstruction after four weeks of therapy. A laparotomy was done with excision and adhesiolysis leading to relief of symptoms. A histology report revealed the lesion to be Kaposi's sarcoma. Our patient was diagnosed to be having immune reconstitution inflammatory syndrome associated with AIDS-associated gastrointestinal limited Kaposi's sarcoma, which presented as acute intestinal obstruction. Our patient was treated with paclitaxel post-operatively and HAART was continued. Our patient responded to therapy.Immune reconstitution inflammatory syndrome involving Kaposi's sarcoma may occur in HAART-naïve individuals with AIDS-related Kaposi's sarcoma. Gastrointestinal Kaposi's sarcoma may present with sudden increase in size or inflammation leading to acute intestinal obstruction. This does not indicate failure of HAART or a need for changes in anti-retroviral regimen. Kaposi's sarcoma (KS) remains the most common tumor in individuals infected with human immunodeficiency virus (HIV) and is associated with significant morbidity and mortality. Highly active anti-retroviral therapy (HAART) for acquired immunodeficiency syndrome (AIDS) decreases the incidence of KS, prolongs the time to treatment failure in KS, leads to resolution of individual lesions, and also decreases KS herpes virus viral load. Immune reconstitution inflammatory syndrome (IRIS) is well recognized as a complication of using HAART for treating AIDS, especially when associated with mycobacterial, fungal or viral infection. Our case demonstrates that KS can worsen during HAART-associated increases in the cluster of differentiation 4 (CD4) count and KS is an IRIS-associated disease. IRIS-KS may be associated with significant complications. It is important for clinicians to realize that KS-associated IRIS does not indicate failure of HAART or a need for changes in the anti-retroviral regimen. Instead, chemotherapy in conjunction with HAART can effectively control the symptoms of IRIS as well as resolve KS.A 36-year-old Asian Indian male patient attended our clinical immunology outpatient clinic with a fever of two months duration and two episode of herpes zoster in the last year. He also complained of significant weight loss in the last three months. He had no history of chronic cough or diarrhea. He was not a diabetic and not alcoholic. Our patient had a history of multiple unprotected sexual exposure around eight years ago.3. He was put on HAART therapy consisting of nevirapine 30 mg twice a day, lamuvidine 150 mg twice a day and zidovudine 300 mg twice a day.On clinical examination his vitals were normal. He had a mild pallor and oral candidiasis. He had no significant lymphadenopathy or any cutaneous lesions. A systemic examination revealed no significant abnormality. Routine investigations were within normal limits. An ultrasound of his abdomen revealed no abnormality. An enzyme-linked immunosorbent assay (ELISA) for HIV-1 was positive and this was confirmed by western blot. Serology for Hepatitis B and C was non-reactive. Our patient's CD4 count was 67/mmAfter 12 days our patient came back with a complaint of upper abdominal discomfort. An abdominal examination revealed mild epigastric tenderness, no organomegaly and normal peristaltic sound. An ultrasound of his abdomen was normal. He was presumed to be suffering from gastrointestinal side-effects, which is very common during initiation of HAART, and advised to take a proton pump inhibitor.3. We put our patient on conservative management with fluids and nasogastric suction. However there was no improvement in 24 hours and our patient complained of increasing pain. A surgical consultation was taken and an exploratory laparotomy was performed with universal precautions. On exploration, small bowel loops were seen matted with a minimal amount of ascites. Purple colored patches, three in number, were seen on the serosal surface of his small bowel. Three thick walled, purple colored rounded cystic lesions adherent to surrounding bowel loops and omentum containing dirty white fluids were seen on the small bowel mesentery. The lesions had prominent veins over their surfaces  and the HAART was continued along with supportive management. He responded well to treatment and is doing well on follow-up.Our patient's symptoms improved after surgery. He was started on an oral diet after five days. The histological examination of the excised lesion showed tortuous blood vessels lined by a single layer of pleomorphic endothelial cells with extra-vasation of red blood cells in the stroma with spindle cells Figure  suggestiKS is a multi-focal neoplastic disease that originates from the lymphatic endothelium, most frequently involving the skin. Also commonly involved are the mucous membrane, lymph nodes, gastrointestinal system and lungs. Lesions have been reported in virtually every organ including the heart and central nervous system. Human herpes virus-8 has been strongly implicated as a co-factor in the pathogenesis of KS. In HIV-infected patients, KS is an AIDS-defining illness.Gastrointestinal involvement in KS is frequent in patients with advanced HIV disease. In one series of KS, gastrointestinal lesions were found in up to 51% of the cases . In anotGastrointestinal involvement commonly occurs in association with cutaneous lesions or lymph node involvement, with gastrointestinal tract involvement alone occurring in only 3.5% of cases . GastroiIRIS is an inflammatory reaction to an opportunistic pathogen and/or tumor antigen that occurs early after initiation of HAART in patients with AIDS and is temporally related to an increase in the host's CD4+ lymphocyte count [Initiation of HAART is usually associated with a regression of KS. However in this case there was probably a rapid increase in the size of the KS lesion, causing intestinal obstruction. Several features of this case suggest that the worsening symptoms and clinical finding represented IRIS rather than progressive KS. The rise in his CD4 count and the temporal relationship of bowel obstruction to HAART initiation also support the diagnosis of KS-IRIS.Although KS is prevalent among HIV-1 infected persons, IRIS during anti-retroviral treatment of AIDS-associated KS has only been reported three times -7. In onet al. identified 150 therapy-naïve patients with a first presentation of KS and recorded their clinic-pathologic features prospectively[In a review of 5,832 patients with AIDS undergoing HAART, Bower pectively. They idThis is a rare case of IRIS associated with AIDS-related gastrointestinal limited KS, presenting as an acute intestinal obstruction. It is likely that KS-associated IRIS is more common than the literature reflects due to limited awareness of this condition. It is important for clinicians to realize that KS-associated IRIS does not indicate failure of HAART or a need for changes in the anti-retroviral regimen. Instead, chemotherapy in conjunction with HAART can effectively control the symptoms of IRIS as well as resolve KS, especially when KS-IRIS is severe or there is visceral involvement.IRIS-KS may occur in HAAR- naïve individuals with AIDS-related KS. Gastrointestinal KS may present with a sudden increase in size leading to acute intestinal obstruction. KS-associated IRIS does not indicate failure of HAART or a need for changes in the anti-retroviral regimen.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journalThe authors declare that they have no competing interests.JP and AS were involved in patient care and management. They also conceived the report and were involved in preparation of the manuscript. DKS was involved in the surgery and patient management. HSK helped diagnose our patient and review the manuscript. DG was involved in counseling and administration. All authors read and reviewed the manuscript.


The techniques for reconstruction of the posterior cruciate ligament (PCL) are still being developed. There are some options when choosing a graft for PCL reconstruction. The bone tendon bone (BTB) and semitendinosus and gracilis tendons (STG) are widely used, but both have advantages and disadvantages. Each result after ligament reconstruction has been reviewed. However, few studies, in which the same surgeon has conducted the same rehabilitation program using the same bone tunnel, have compared postoperative results between BTB and STG. It remains to be clarified whether the results differ according to graft materials and their fixation between the two techniques -5.We performed anterolateral single bundle reconstruction of the PCL using BTB and STG in a total of 30 cases, and the courses of the patients were observed for over one year. The postoperative results of PCL reconstruction using BTB and STG were compared and studied retrospectively. The purpose of the present study was to clarify the features of the two surgical methods and to explore the problems associated with these procedures and measures that could be used to improve them.Thirty patients who had undergone PCL reconstruction and had been followed-up for more than twelve months were the subjects of this study. Our retrospective study included 14 patients treated with the transtibial technique using bone patellar tendon bone (BTB group) and 16 patients who underwent the transtibial anterolateral single-bundle technique using semitendinosus and gracilis tendons (STG group).The study population comprised 13 men and 1 woman in the BTB Group, and 12 men and 4 women in the STG Group. The mean length of follow-up was 35.0 ± 40.0 months (12–156) in the BTB group and 23.5 ± 14.7 months (12–62) in the STG group. The mean time from injury to surgery was 24.1 ± 33.3 months (2–125) in the BTB group and 23.4 ± 37.5 months (1–144) in the STG group. At the time of the operation, the mean age was 31.1 ± 7.7 years (19–43) in the BTB group and 35.1 ± 9.4 years (19–54) in the STG group. The cause of injury included 4 motor vehicle accidents, 6 sports injuries, and 4 accidental fall in the BTB group and 7 motor vehicle accidents, 3 sports injuries, and 6 accidental falls in the STG group. Other associated ligamentous injuries; i.e., posterolateral corner laxities (6 cases), were treated with grafts from the iliotibial tract (2 cases) or hamstrings tendons (4 cases) . Patients with Grade 1 injuries have a palpable but diminished step off. Patients with Grade 2 injuries have lost their step off, but their medial tibia plateau cannot be pushed beyond the medial femoral condyle. Patients with Grade 3 injuries have lost their medial step off, but their medial tibial plateau can be pushed beyond the medial femoral condyle . Statistical analysis was performed using the unpaired t - test (2-tails).The KT-2000 arthrometer was used to evaluate the side-to-side difference of anteroposterior laxity at 30 degrees of flexion in the BTB group(8 cases) and the STG group(10 cases).BTB group: The patients underwent an arthroscopically assisted anterolateral single-bundle PCL reconstruction. A transtibial guide pin was placed slightly lateral of the tibial footprint from the anterolateral cortex of the proximal part of the tibia. Its position was verified with intraoperative fluoroscopy. Then, a tibial tunnel of 10 mm in diameter was drilled. A femoral guide pin was inserted from the medial epicondyle at the two o’clock position in the right knee, 8 to 9 mm proximal to the articular junction. A femoral tunnel of 10 mm in diameter was then created. A bone patellar tendon bone (BTB) autograft of 9 mm in diameter was passed through the tibial tunnel and into the femoral tunnel. Aperture fixation of a bone plug was performed as close as possible to the exit of the tibial bone tunnel. At the time of the graft fixation, Knee position is 10 degrees in flexion. The graft was tensioned with manual maximum stress and fixed with cannulated interference screws  to the femoral and tibial sides with the outside-in technique.STG group: Tibial and femoral tunnels were made in the same manner as described for the BTB group. Suture disc was attached to the quadrupled hamstring tendon autograft for femoral fixation. The prepared graft of 9 or 10 mm in diameter was pulled through the femoral tunnel and into the tibial tunnel. The graft was then tensioned with manual maximum stress and fixed with the post screw technique to the tibia. Knee position is 10 degrees in flexion same as BTB group.The rehabilitation program and weight bearing period were the same for the 2 groups. A knee brace was applied postoperatively with the knee in full extension. A functional PCL brace was fitted at 2 to 12 postoperative weeks. Partial weight bearing using 2 crutches was allowed from the next operative day. Weight bearing was gradually increased to full weight bearing at 3 weeks. Full ADL was allowed from 3 months. Jogging and low impact sports began at 6 months. A full return to sports was allowed from 8 to 12 months depending on the patient.Written informed consent was obtained from the patient for publication of this report and any accompanying images.In the BTB group, on the Posterior drawer test, 7 knees (50%) were Grade 0, and one knee each was Grade 2 and 3, respectively. In the STG group, only 2 knees were Grade 0, and all of the others were Grade 1.In the BTB group, favorable stability was achieved in half of cases, but unfavorable cases were also present. In the STG group, slight elongation was noted in all cases, but none of it was markedly unfavorable  who simultaneously underwent reconstruction with modified Larson method showed improvement from 33.5% before surgery to 52.4% after surgery on stress X-ray radiographic measurement, there was no difference from the patients with PCL reconstruction alone.Typical case 1 (BTB group): A 48-year-old male was injured in a traffic accident. His clinical characteristics were as follows: posterior drawer test, Grade 2; midpoint displacement rate, 38.5%; and Lysholm score, 65 points. At 28 months after surgery, his results for the above parameters were Grade 1, 50.2%, and 93 points, respectively : A 35-year-old female was injured by a fall. Her clinical characteristics were as follows: posterior drawer test, Grade 2; midpoint displacement rate, 32.5%; and Lysholm score, 60 points. At 17 months after surgery, her results for the above parameters were Grade 1, 51.0%, and 87 points, respectively  and allografts (Achilles tendon and BTB). The approaches were classified into single and double bundles, and graft placements were classified into the tibial tunnel and tibial inlay methods . In JapaRegarding the approach, Kohen et al., systematically reviewed biomechanical and clinical studies of PCL reconstruction involving single and double bundles and concluded that at present it is unclear whether its outcomes are superior to those of previous methods .Since Berg et al . first rTherefore, we performed conventional anterolateral transtibial single-bundle reconstruction of the PCL using BTB and STG until now, so we compared the outcomes of both methods with the aim of minimizing complications.When the BTB is used as the graft source, the fixation force is strong because the graft is fixed bone-to-bone with interference screw, which is advantageous; however, it can cause the following problems: muscle weakness of the graft-harvest region, pain around the patellofemoral joint, and wear of the graft caused by sharp angulation at the posterior opening of the tibial tunnel (killer turn). Regarding STG, hamstring tendon grafts can be harvested easily and the soft tissue end of the graft allows it to be easily passed through the tunnel, but graft fixation is inferior, the tendency for graft elongation over time has been reported. The same risk of killer turn is present also in the use of the STG graft, but STG involves eight bundles, and, hence, pressure is estimated to be dispersed even if the angle suddenly changes.In the BTB group, the grade was unchanged in 2 cases: a Grade-3 case and a Grade-2 case, suggesting the presence of re-rupture due to wearing or elongation of the graft. However, the other outcomes of the BTB group were relatively favorable.Therefore, we have attempted to minimize the problems of killer turn at the tibial tunnel by creating an anterolateral tibial bone tunnel, chamfering a tunnel exit as much as possible, preparing a cylindrical bone block into a column in order to fill the space between the graft and tunnel wall, and placing the bone fragment close to the posterior opening of the tibial tunnel ,15.In this study, the subjects included patients who simultaneously underwent treatment for posterolateral corner laxities, but there was no difference in the outcome between these patients and those who underwent PCL reconstruction alone.Since the study was not prospective, short follow-up period, and the number of patients was small, precise comparison of the outcome is impossible at present, and so further evaluation is necessary.1. We compared the clinical results of two groups treated with anterolateral single-bundle reconstruction of the posterior cruciate ligament using the BTB or STG method with short term follow-up(a minimum of 12 months).2. Post-operative outcome of the BTB group was relatively favorable except 2 cases. Excellent stability and fair results were obtained in the BTB group. The STG group showed slight residual knee laxity in all cases, but there were no complete failures.3. Several techniques have been advocated to minimize the problem of killer turn in the BTB method.The authors declare that they have no competing interests.YM drafted the manuscript. KS, TB and KK contributed to study design and manuscript sturucture. All authors read and approved the final manuscript.1Department of Orthopaedic Surgery, Juntendo University Urayasu Hospital, 2-1-1 Tomioka, Urayasu, Chiba prefecture Postal code 279-0021, Japan.2Department of Orthopaedic Surgery, Juntendo University School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo Postal code 113-8421, Japan.


In addition, the slice co-culture system revealed that the NMDA receptors and the medial prefrontal cortex play an essential role in the dopaminergic sensitization. This in vitro sensitization model provides a unique approach for studying mechanisms underlying behavioral sensitization to drugs of abuse.Repeated intermittent exposure to psychostimulants and morphine leads to progressive augmentation of its locomotor activating effects in rodents. Accumulating evidence suggests the critical involvement of the mesocorticolimbic dopaminergic neurons, which project from the ventral tegmental area to the nucleus accumbens and the medial prefrontal cortex, in the behavioral sensitization. Here, we examined the acute and chronic effects of psychostimulants and morphine on dopamine release in a reconstructed mesocorticolimbic system comprised of a rat triple organotypic slice co-culture of the ventral tegmental area, nucleus accumbens and medial prefrontal cortex regions. Tyrosine hydroxylase-positive cell bodies were localized in the ventral tegmental area, and their neurites projected to the nucleus accumbens and medial prefrontal cortex regions. Acute treatment with methamphetamine (0.1–1000 µM), cocaine (0.1–300 µM) or morphine (0.1–100 µM) for 30 min increased extracellular dopamine levels in a concentration-dependent manner, while 3,4-methylenedioxyamphetamine (0.1–1000 µM) had little effect. Following repeated exposure to methamphetamine (10 µM) for 30 min every day for 6 days, the dopamine release gradually increased during the 30-min treatment. The augmentation of dopamine release was maintained even after the withdrawal of methamphetamine for 7 days. Similar augmentation was observed by repeated exposure to cocaine (1–300 µM) or morphine (10 and 100 µM). Furthermore, methamphetamine-induced augmentation of dopamine release was prevented by an NMDA receptor antagonist, MK-801 (10 µM), and was not observed in double slice co-cultures that excluded the medial prefrontal cortex slice. These results suggest that repeated psychostimulant- or morphine-induced augmentation of dopamine release,  Psychostimulants (e.g. amphetamines and cocaine) and opiates (e.g. morphine) share the ability to cause drug dependence and addiction. In rodents, repeated intermittent exposure to psychostimulants and morphine leads to progressive augmentation of their locomotor activating effects. This phenomenon, termed behavioral sensitization, is thought to underlie certain aspects of drug addiction N-methyl-d-aspartate (NMDA) and non-NMDA receptors, which are innervated from the limbic and cortical areas, such as the mPFC, hippocampus and amygdala to the NAc The effect of amphetamines on dopamine release is mainly attributed to their binding to and reversal of dopamine transporter (DAT) function, resulting in both reuptake inhibition and release of dopamine in vivo experiments in whole animals, because these addiction-related phenomena are thought to be due to long-term alterations in psychological behavior caused by synaptic plasticity in the mesocorticolimbic dopaminergic neurons. Acute effects of drugs of addiction on dopaminergic function have been extensively assessed by in vitro experiments using cell lines expressing DAT in vitro culture systems that recapitulate cell-to-cell interactions between different parts of the brain are anticipated to provide much valuable data about the long-term effects of drugs, facilitating investigations of the neural plasticity underlying drug addiction.Many aspects of drug addiction have been assessed by 1-like, but not D2-like, dopamine receptors To this end, Maeda et al. reconstructed the mesocorticolimbic system using rat triple organotypic slice co-cultures of the mesencephalic slice including the VTA, the ventral striatal slice including the NAc, and the mPFC slice in vitro, we examined the effects of acute and chronic effects of psychostimulants and morphine on dopamine release. Here we report that repeated exposure of the triple slice co-cultures to methamphetamine (METH), cocaine or morphine augmented their dopamine releasing effects, i.e. dopaminergic sensitization. Furthermore, we examined the involvement of NMDA receptors and the mPFC in the dopaminergic sensitization.To explore the utility of the triple slice co-cultures for studying certain processes relevant to behavioral sensitization To assess the reconstruction of the mesocorticolimbic dopaminergic neurons in the VTA/NAc/mPFC triple slice co-cultures, immunostaining was performed for tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines . TH-posiF5,22 = 11.22, p<0.001, cocaine; F5,40 = 3.857, p<0.01, morphine; F4,13 = 15.21, p<0.001). Significant increases were observed by METH at concentrations of 100 and 1000 µM (p<0.001), cocaine at concentrations of 100 and 300 µM  and morphine at a concentration of 100 µM (p<0.001), compared with phosphate-buffered saline (PBS)-treated triple slice co-cultures. Single treatment with 3,4-methylenedioxymethamphetamine  for 30 min tended to increase the extracellular dopamine level, although this increase was not significant .The effects of single treatment of the VTA/NAc/mPFC triple slice co-cultures with METH, cocaine, MDMA or morphine on dopamine release were examined . Single F5,25 = 5.007, p<0.01) and drug effects  were observed as well as a day×drug significant interaction . On day 7, both repeated METH- and PBS-exposed co-cultures were challenged by METH at a concentration of 10 µM for 30 min (p<0.01). The effect of withdrawal of METH on the repeated METH induced-augmentation of dopamine release was examined .To examine the effect of repeated exposure to METH on dopamine release, the VTA/NAc/mPFC triple slice co-cultures were exposed to METH at a concentration of 10 µM in Krebs-Ringer-Henseleit (KRH) buffer for 30 min every day for 6 days . The extr 30 min . The METexamined . When MEF4,21 = 8.953, p<0.001). Repeated METH exposure displayed a bell-shaped curve of concentration-dependence; the METH challenge-evoked dopamine releases following the repeated METH exposure at concentrations of 100 and 1000 µM were lower than those following the repeated METH exposure at a concentration of 10 µM. Compared with the slice co-cultures repeatedly exposed to PBS, a significant increase in the extracellular dopamine level was observed in the slice co-cultures repeatedly exposed to METH at a concentration of 10 µM (p<0.001).Next, we examined the concentration-dependence of the repeated METH induced-augmentation of dopamine release . The triF4,20 = 3.514, p<0.05 and F2,14 = 4.572, p<0.05, respectively) in a concentration-dependent manner. Compared with co-cultures repeatedly exposed to PBS, significant increases in the extracellular dopamine level were observed by the repeated exposure to cocaine at a concentration of 300 µM (p<0.05) and morphine at a concentration of 100 µM (p<0.05).The triple slice co-cultures were exposed to cocaine (1–300 µM) or morphine (10 and 100 µM) for 30 min every day for 6 days, and then the co-cultures were challenged with cocaine (10 µM) or morphine (100 µM), respectively, in KRH buffer for 30 min on day 7 . Repeatep<0.05).To investigate whether NMDA receptors are involved in the induction of the augmentation of dopamine release following the repeated exposure to METH, we examined the effect of the NMDA receptor antagonist MK-801 . The VTAp<0.001).Next, to examine the role of mPFC region in the augmentation of dopamine release following repeated METH exposure, the slice co-cultures composed of the mesencephalic and ventral striatal slices were used without the mPFC slices . After rin vivoin vivo physiology and allowed innervation of the mesocorticolimbic dopaminergic neurons. On the other hand, the mPFC is shown to connect to the NAc through the corticoaccumbens glutamatergic pathway To analyze the mechanisms underlying drug sensitization, we reconstructed the mesocorticolimbic system using rat organotypic triple VTA/NAc/mPFC slice co-cultures, as previously reported by Maeda et al. in vivo is mediated through indirect mechanisms via MDMA-induced serotonergic activation and direct DAT reversal In the triple slice co-culture, single treatment with METH and cocaine caused dopamine release in a concentration-dependent manner. METH directly acts on DAT located on dopaminergic nerve terminals and reverses dopamine reuptake, resulting in both reuptake inhibition and release of dopamine. In contrast, cocaine only blocks reuptake of dopamine at dopaminergic nerve terminals in vitro systems, namely cell lines expressing DAT However, the concentrations required for the dopamine release induced by single treatment with METH, cocaine and morphine in the triple slice co-cultures are higher than those reported in other in vivo studies have indicated that repeated intermittent exposure to psychostimulants and morphine leads to the augmentation of dopamine release from the mesocorticolimbic dopaminergic terminals in the NAc and mPFC, which is thought to be the major cause of the behavioral sensitization . Morphine hydrochloride and cocaine hydrochloride were purchased from Takeda Chemical Industries . MDMA hydrochloride was a gift from Dr. Tatsunori Iwamura . (+)-MK-801 hydrogen maleate, an NMDA receptor antagonist, was purchased from Sigma . Drugs were dissolved in PBS, and then diluted in the culture medium.l-glutamine, was supplied at a volume of 0.7 ml per well. The triple slice co-cultures were maintained at the liquid/air interface for 18 days in an incubator at 37°C in a 5% CO2 humidified atmosphere, and subsequently used in experiments. The culture medium was exchanged for fresh medium on the day following culture preparation, and on every second day thereafter.All animal care and experimental procedures were approved by the Kyoto University Animal Research Committee. Organotypic slice co-cultures were prepared according to the methods described previously Slice co-cultures were fixed with 0.1 M phosphate buffer containing 4% paraformaldehyde and 4% sucrose for 2 hr. After washing with PBS, fixed cultures were permeabilized and blocked with PBS containing 0.2% Triton X-100 and 10% fetal calf serum for 1 hr. The cultures were then incubated overnight at 4°C with rabbit anti-TH polyclonal antibody , and washed with PBS three times. The cultures were incubated for 1 hr in a secondary antibody solution containing either Alexa Fluor 488 goat anti-rabbit IgG , or biotinylated anti-rabbit IgG  and washed with PBS three times.2O2. Specimens were dehydrated through a graded ethanol series and mounted on glass slides with glycerol for observation with a microscope using bright-field illumination.For immunofluorescent staining, the cultures were transferred to glass slides and cover-slipped using Vectashield hard-set mounting medium (Vector Lab). Immunofluorescence was visualized using a Nikon Diaphot 200 microscope equipped with a laser scanning confocal imaging system . For immunostaining using the biotin-avidin peroxidase method, cultures were treated for 1 hr with avidin–biotinylated horseradish peroxidase complex  at room temperature. After a further wash with 50 mM Tris-buffered saline, peroxidase was visualized with 0.07% 3,3′-diaminobenzidine and 0.018% H+/K+-ATPase , followed by probing with horseradish peroxidase-conjugate secondary antibody. Proteins were visualized by Immobilon Western HRP substrate  and detected by X-ray film.After 18 days culture, slice co-cultures were separated into VTA, NAc and mPFC parts under dissecting microscope, and these parts from 4 culture inserts were harvested in 100 µL Lysis buffer  and 1% protease inhibitor cocktail ). After harvest, these parts were homogenized, sonicated and centrifuged at 600 g for 5 min, and protein concentration of resulting supernatants were measured by the bicinchoninic acid protein assay . Equal volume of 2× Laemmli sample buffer were added to supernatants and stored at −20°C until use. Thawed samples were analyzed by SDS-PAGE, transferred to polyvinylidene difluoride membrane and probed with anti-dopamine transporter antibody  or anti-Na2, 1.2 mM CaCl2, 10 mM D-glucose, 15 mM HEPES, 5 mM HEPES-Na, 0.2 mM ascorbic acid; pH 7.4) three times. After the washes, the inserts were preincubated in KRH buffer for 15 min, then transferred to 0.7 mL KRH buffer containing drugs and incubated for 30 min. After incubation, KRH buffer was collected into vials, and acid solution was added that contained 1 M HClO4, 100 mM Na2S2O5 and 10 mM EDTA. To quantify dopamine concentration, the collected aliquot (20 µl) was automatically injected and analyzed by high performance liquid chromatography with an electrochemical detection system . Dopamine was quantified by reference to a linear calibration curve ranging from 0.1 to 100 nM. The detection limits for dopamine were estimated to be approximately 10 pM per 20 µl sample.Measurement of extracellular dopamine levels was performed as previously described t-test. Data with more than two groups were compared by one-way analysis of variance (ANOVA), followed by the Dunnett's multiple comparison test. The time-course data was analyzed by two-way ANOVA for repeated measures, followed by the Bonferroni post-hoc test. Differences of p<0.05 were considered statistically significant.Data are presented as the mean ± S.E.M. Differences between two groups were compared using the Student's Figure S1Effect of sustained exposure to METH on the propidium iodide (PI) uptake in the VTA/NAc/mPFC triple slice co-cultures. The triple slice co-cultures were exposed to PBS, METH (1–1000 µM) or NMDA (300 µM) in the presence of PI (5 µg/ml). After 2 days incubation, the PI fluorescence of each slice was observed with an inverted fluorescence microscope with a rhodamine filter set. The triple slice co-cultures exposed to N-methyl-d-aspartate  for 2 days were used to determine the degree of the standard injury. Representative photomicrographs of PI fluorescence in the NAc (upper), mPFC (middle) and VTA (bottom) regions, respectively, are shown. Sustained exposure of the slice co-cultures to METH for 2 days exhibited concentration-dependent increases in PI fluorescence in each of the NAc, mPFC, and VTA regions. In all regions, a small number of PI positive cells was observed with 10 µM METH, and marked increases of PI positive cells were observed with 100 and 1000 µM METH. The total number of PI positive cells in the mPFC region was greater than in the other two regions. This is likely due to the fact that in the mPFC region, there were a greater total number of cells in the original triple slice co-cultures.(TIF)Click here for additional data file.


Molecular imaging is an emerging field that integrates advanced imaging technology with cellular and molecular biology. It can realize noninvasive and real time visualization, measurement of physiological or pathological process in the living organism at the cellular and molecular level, providing an effective method of information acquiring for diagnosis, therapy, and drug development and evaluating treatment of efficacy. Molecular imaging requires high resolution and high sensitive instruments and specific imaging agents that link the imaging signal with molecular event. Recently, the application of new emerging chemical technology and nanotechnology has stimulated the development of imaging agents. Nanoparticles modified with small molecule, peptide, antibody, and aptamer have been extensively applied for preclinical studies. Therapeutic drug or gene is incorporated into nanoparticles to construct multifunctional imaging agents which allow for theranostic applications. In this review, we will discuss the characteristics of molecular imaging, the novel imaging agent including targeted imaging agent and multifunctional imaging agent, as well as cite some examples of their application in molecular imaging and therapy. Molecular imaging is a rapidly developed multidiscipline which involves molecular biology, chemistry, computer, engineering, and medicine . It can Radionuclide molecular imaging including PET and SPECT is the earliest and most mature molecular imaging technique. Due to its advantages of high sensitivity and quantifiability, radionuclide molecular imaging plays an important role in clinical and preclinical researches . Over th11C, 15O, 18F, and 131I) that are intravenously injected into the body. These isotopes emit positrons which are ejected from the nucleus as a result of springless interactions with electrons in surrounding tissue. The positrons rapidly lose kinetic energy by spreading around the tissue and collide with an electron to form two 511 keV photons which are taking trajectory 180° apart, and this is an event known as annihilation [PET is the molecular imaging modality most extensively used in current clinic routine. It measures the signal originated from the radioactive decay of neutron-deficient radioisotopes , SPECT can distinguish different radioisotopes, therefore making it possible to image different targets simultaneously. Hijnen et al. [99mTc/123I SPECT, which proved that simultaneous 99mTc/123I imaging can provide images of similar quantitative accuracy through GSFA as when using sequential and scatter-free99mTc/123I imaging in brain SPECT.Unlike PET, SPECT directly detects gamma-ray photon emitted by the chosen radionuclide during their decay. Compared with PET, SPECT is more affordable and extensively employed in the clinical routine, but it is generally less sensitive since the photons which are not traveling along the axis of the collimator are rejected by the scanner. The spatial resolution (8–10 mm) of SPECT is lower than that of clinical PET (5–7 mm) , but then et al.  took advαvβ3 integrin and MR molecular imaging was a promising strategy for evaluation of colorectal cancers associated angiogenesis.Magnetic resonance imaging (MRI) is a highly versatile imaging modality . During −2~10−3 mol/L), CT is always combined with other imaging modalities such as SPECT, PET to provide anatomical parameters for the biochemical and physiological findings [CT imaging technologies have undergone a very fast development in the last years. High resolution small animal CT (micro-CT) has transformed CT imaging from organ, tissue to molecular level, which is playing an increasingly important role in preclinical researches , 30. Thefindings . Recentlfindings . For exafindings  reportedfindings  found thfindings  conjugatOptical molecular imaging technology is an emerging technology, based on genomics, proteomics, and modern optical technology. At present, the most widely used optical molecular imaging modalities in vivo include bioluminescence imaging (BLI) and fluorescence imaging. As optical imaging is performed through the use of light, thus it is considered as relatively safe. And due to their advantages of high sensitivity and low cost, optical imaging plays a central role in the investigation of tumor occurrence, progressions and relevant drug development , 37. TheBLI is taking advantage of the light produced by the enzymatic oxidation reaction of luciferase and its substrate. The luciferase is usually generated from the reporter gene. As opposed to fluorescence imaging, the light source of BLI is from interior. And since tissue do not produce endogenous bioluminescence, the sensitivity and signal to background is better than fluorescence imaging. BLI is widely used to observe the biological processes in vivo like disease progression, certain gene expression, tumor growth, and metastasis and evaluate the effect of certain treatments. For example, Niu et al.  introducαvβ3 and vascular endothelial growth factor receptor (VEGFR) are overexpressed in tumor; they used the integrin αvβ3 fluorescent probe to detect the tumor angiogenesis in nude mice. The result showed there were strong fluorescence signals for αvβ3 in vivo via a fluorescent scanner, which indicated angiogenesis increased in the cancer tissue. Also, they proved that the targeted visualization of VEGF could be achieved via a fluorescent-labeled antibody against the VEGFR . Hence, fluorescence imaging is mainly used in animal researches. Atreya et al.  took advhe VEGFR .With the use of ultrasound contrast agent, ultrasound imaging enables specific and sensitive depiction of molecular targets , 41. ComBesides, for diagnostic purposes, molecular ultrasound imaging can also be applied for theranostic purposes. This is mainly achieved by loading gene or drug onto microbubble. The interaction between ultrasound and microbubble results in stable and inertial cavitation, which can transiently increase vascular and cellular membrane permeability, thereby potentially augments the extravasation and/or take up of drugs or genes loaded on microbubble. Li et al.  prepared18F-FDG accumulation on the PET image with precise correlation to anatomical findings' locations on the CT scan, thus the specificity and sensitivity of PET in detecting lesion are enhanced, so as the accuracy in delineating target volume. Abgral et al. [18F-FDG PET/CT in these patients. The results showed that the sensitivity, specificity, and accuracy of 18F-FDG PET/CT for the diagnosis of HN cancer recurrence were 100%, 85%, and 90%, respectively. The positive predictive value was 77%, while the negative predictive value was 100%.Among all molecular imaging techniques, every molecular imaging technique has its advantages and disadvantages. No single one is perfect and enough to provide comprehensive information for disease diagnosis . In genel et al.  conducteMRI shows obvious advantages over CT including excellent soft tissue contrast, high spatial resolution, and no ionizing radiation; thus studies of PET/MRI become the focus of concern and have achieved initial progress. Lee et al.  used PETIn addition to the combination of PET and CT or MRI, other multimodality imaging methods have been reported, such as OI/US, OI/CT, OI/MRI, and US/MRI –58. OI iTherefore, it can be seen that multimodality imaging is not a simple addition of various imaging technologies but rather for producing synergistic effect. It can provide more information to understand the biological processes comprehensively and objectively. But it is still difficult to carry out multimodality imaging due to existing problems regarding the accuracy of coregistered image, extra ionizing radiation, the extra dosage of contrast agent, and the toxicity of fused contrast agents. Therefore, it is in urgent need of developing multimodality molecular imaging agent.Molecular imaging depends greatly on the development of specific and sensitive imaging agents, which is a pivotal rate-limiting step in the development of molecular imaging . In a mo18F-FDG is the most widely used imaging agent based on small molecules, which is clinically applied for cancer imaging [Small molecules, of which the size is usually less than 500 Da, are playing an important role in molecular imaging. Due to their small size, small molecules have a wide range of application including intracellular and central nervous system.  imaging . Ren et  imaging  reported18F labelled 2 cystine knot peptides and performed micro-PET on BxPC3 pancreatic adenocarcinoma xenografts in mice with them. Results indicated that these cystine knot peptide tracers had high tumor uptake, showing translational promise for cancer imaging.Peptide is an important class of ligand used for molecular imaging . CompareCharacterized by the ability to bind their target with high specificity and affinity and easy synthesis, antibodies have been applied for diagnosis and therapy . So far,67Ga radionuclide imaging and MRI. The SPECT image showed that this probe exhibited a better accumulation in tumour 24 h after injection than the control group. Furthermore, MRI images showed the AS1411 conjugated nanoparticle as black signal in tumours 24 h after injection, while there were no T2 negative signals in the control group. Finally, fluorescence images exhibited a higher signal in tumours of mice injected with aptamer conjugated nanoparticles compared to the control one. Taken together, these results proved that aptamers can deliver the nanoparticles to tumor tissue  [With multimodality imaging techniques clearly on the rise, the development over these new techniques has led to explosive growth in multimodal imaging agent researches , 75. Sinte (SFB) . Hence, Apart from imaging agent and ligands, therapeutic drug or gene can also be incorporated in nanoparticles to construct theragnostic agent which have an important role in therapy. This agent enables observation of the extent of disease prior to therapeutic intervention. The ability to identify disease status and controlled delivery of drug using the same agent would help ensure that only potential responsive patients would be treated. Therefore, theragnostic agents show a promising prospective in therapy. Yan et al.  have synMolecular imaging technology truly enables dynamical, quantitative visualization of specific biochemical activity without trauma in vivo at cellular and molecular level. It has a direct impact on the modern and future medicine. In recent years, molecular imaging technology has seen certain progresses in the early diagnosis, curative effect monitoring of diseases, drug development, gene therapy, and other fields, but some key problems regarding theory, technology, and system, especially molecular imaging agents and imaging equipment, are not solved yet. The development of a new probe is not straightforward. Due to barriers in delivery, biological compatibility, and the diversity between species, there are only a few of clinical molecular imaging agents available currently. With the development of technology, it is expected that more advancement will be achieved in the area of molecular imaging agent including targeted molecular imaging agent and multifunctional molecular imaging agents. The application of nanoparticles has provided a platform for theranostic researches.Additionally, although a number of molecular imaging instruments are available, all have their limitations. For instance, MRI is a useful clinical diagnostic tool, but it suffers from somewhat poor sensitivity. Therefore, more focus should be performed on refining and improving upon existing instrumentation and further efforts geared toward the combination of different modalities so as to make up the disadvantages of different instruments. Multimodality imaging has many advantages, but some problems still exist and need to be solved, such as the difficulty of designing a PET/MRI system suited for the entire body, cost increase, performance improvement, and multimodal contrast agent fusion.Furthermore, as an emerging interdisciplinary science which brings together nuclear medicine, ultrasonic medicine, radiology, pharmacy, and materials science, the development of molecular imaging needs to strengthen interdisciplinary cooperation. Awareness of importance of multidisciplinary joint research should be strengthened.The rapid expansion of molecular imaging application shows a promising prospect. Although overall the molecular imaging is still at the initial stage of development, we believe that within the support and cooperation from imaging experts and scholars, molecular imaging techniques would eventually realize clinical transformation.