Anatomical Landmark Technique for Femoral Tunnel Placement of Lateral Extra-Articular Tenodesis.

The anterolateral ligament is a crucial part of the anterolateral complex of the knee, providing rotator stability to the knee and being a primary restraint to tibial internal rotation. Lateral extra-articular tenodesis added to anterior cruciate ligament reconstruction can reduce pivot shift without sacrificing the range of motion or increasing the risk of osteoarthritis. A 7- to 8-cm longitudinal skin incision is made and a 9.5- to 10-cm × 1- cm wide iliotibial band graft is dissected, leaving the distal attachment intact. The free end is whip stitched. One of the most important steps during the procedure is the identification of the site of attachment of the iliotibial band graft. The leash of vessels, fat pad, lateral supracondylar ridge, and fibular collateral ligament serve as important landmarks. The tunnel is drilled from the lateral femoral cortex with a guide pin and reamer pointing 20 to 30° anteriorly and proximally while the arthroscope visualizes the femoral anterior cruciate ligament tunnel. The graft is routed under the fibular collateral ligament. The graft is fixed with a bioscrew while the knee is kept in 30° flexion and the tibia is kept in neutral rotation. We believe that lateral extra-articular tenodesis gives the anterior cruciate ligament graft a good chance for faster healing along with addressing anterolateral rotatory instability. Choosing a correct fixation point is very important to restore normal knee biomechanics.

Factors Associated With Persistence of Dyspnea and Change in Health-Related Quality of Life in Patients With COVID-19 After Discharge.

Introduction Coronavirus disease 2019 (COVID-19) is a deadly virus affecting multiple organ systems, predominantly the respiratory system. Dyspnea along with the deterioration of health-related quality of life (HRQoL) is common in COVID-19 patients discharged from a dedicated Coronavirus disease (COVID) hospital. Very few studies in India used HRQoL for the assessment of COVID-19 patients after discharge. Our article aims to assess the factors associated with the persistence of dyspnea and HRQoL in discharged patients of COVID-19. Methods A total of 48 patients were included in this prospective observational study. Ethical approval from Institutional Ethics Committee was obtained before the enrolment of patients. Patients having dyspnea at exertion and during discharge were selected for this study. Modified Medical Research Council (mMRC) scale and modified Borg scale were used for assessing dyspnea on activity, and Saint George's Respiratory Questionnaire (SGRQ) was used to assess HRQoL. Data were collected on the day of discharge (D0) and after 60 days (D60) post-discharge. The significance of changes in parameters from D0 to D60 was evaluated by paired t-test. Results The mean mMRC, modified Borg, and SGRQ scores at D0 were 2.38±0.98, 3.15±2.12, and 45.36±27.32, respectively, which were improved to 0.94±0.86, 0.94±1.27, and 19.22±18.96 at D60. Age showed significant positive correlations with initial modified Borg (r=0.292, p=0.044) and SGRQ (r=0.332, p=0.021) scores. Body mass index showed significant positive correlations with initial mMRC (r=0.352, p=0.014) and SGRQ (r=0.419, p=0.003) scores. Conclusion Our study showed that on discharge, many COVID patients have impaired HRQoL. Many of them also have dyspnea on exertion. With the early institution of standard pulmonary rehabilitation protocol, symptoms and HRQoL improves rapidly in a month. Different influencing factors were identified. Long-term follow-up with a bigger sample size is needed to formulate a management strategy for these patients.

A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer.

BACKGROUND: We previously showed that the bacterial lipopeptide Pam3Cys-Ser-Ser, meanwhile established as a toll-like receptor (TLR) 1/2 ligand, acts as a strong adjuvant for the induction of virus specific CD8+ T cells in mice, when covalently coupled to a synthetic peptide. CASE PRESENTATION: We now designed a new water-soluble synthetic Pam3Cys-derivative, named XS15 and characterized it in vitro by a TLR2 NF-κB luciferase reporter assay. Further, the capacity of XS15 to activate immune cells and stimulate peptide-specific CD8+ T and NK cells by 6-sulfo LacNAc+ monocytes was assessed by flow cytometry as well as cytokine induction using immunoassays. The induction of a functional immune response after vaccination of a volunteer with viral peptides was assessed by ELISpot assay and flow cytometry in peripheral blood cells and infiltrating cells at the vaccination site, as well as by immunohistochemistry and imaging. XS15 induced strong ex vivo CD8+ and TH1 CD4+ responses in a human volunteer upon a single injection of XS15 mixed to uncoupled peptides in a water-in-oil emulsion (Montanide™ ISA51 VG). A granuloma formed locally at the injection site containing highly activated functional CD4+ and CD8+ effector memory T cells. The total number of vaccine peptide-specific functional T cells was experimentally assessed and estimated to be 3.0 × 105 in the granuloma and 20.5 × 106 in peripheral blood. CONCLUSION: Thus, in one volunteer we show a granuloma forming by peptides combined with an efficient adjuvant in a water-in-oil-emulsion, inducing antigen specific T cells detectable in circulation and at the vaccination site, after one single vaccination only. The ex vivo T cell responses in peripheral blood were detectable for more than one year and could be strongly boosted by a second vaccination. Hence, XS15 is a promising adjuvant candidate for peptide vaccination, in particular for tumor peptide vaccines in a personalized setting.

Spatial variation of trace element concentration and contamination assessment in the coral reef sediments of Lakshadweep Archipelago, Indian Ocean.

Surface sediments were collected from the shore and lagoons of Kavaratti, Kadmat and Agatti islands of Lakshadweep Archipelago and analysed for trace element concentration. The sediment contamination was assessed on the basis of geochemical, biological hazard and ecological risk indices. Except Cd and Pb, all the other trace elements selected for the study were below the contamination level. Compared to Kadmat, Kavaratti and Agatti were more polluted and the pollution was pronounced in lagoons than shore. Population pressure, untreated sewage, diesel based power generation, shipping and tourism activities contribute to sediment contamination. Statistical analysis revealed the association of trace elements with sedimentary characteristics due to anthropogenic sources.

Activated integrins identify functional antigen-specific CD8+ T cells within minutes after antigen stimulation.

Immediate ß2-integrin activation upon T cell receptor stimulation is critical for effective interaction between T cells and their targets and may therefore be used for the rapid identification and isolation of functional T cells. We present a simple and sensitive flow cytometry-based assay to assess antigen-specific T cells using fluorescent intercellular adhesion molecule (ICAM)-1 multimers that specifically bind to activated ß2-integrins. The method is compatible with surface and intracellular staining; it is applicable for monitoring of a broad range of virus-, tumor-, and vaccine-specific CD8+ T cells, and for isolating viable antigen-reacting cells. ICAM-1 binding correlates with peptide-MHC multimer binding but, notably, it identifies the fraction of antigen-specific CD8+ T cells with immediate and high functional capability (i.e., expressing high levels of cytotoxic markers and cytokines). Compared with the currently available methods, staining of activated ß2-integrins presents the unique advantage of requiring activation times of only several minutes, therefore delivering functional information nearly reflecting the in vivo situation. Hence, the ICAM-1 assay is most suitable for rapid and precise monitoring of functional antigen-specific T cell responses, including for patient samples in a variety of clinical settings, as well as for the isolation of functional T cells for adoptive cell-transfer immunotherapies.

Validation of Immunomonitoring Methods for Application in Clinical Studies: The HLA-Peptide Multimer Staining Assay.

BACKGROUND: Validated assays are essential to generate data with defined specificity, consistency, and reliability. Although the process of validation is required for applying immunoassays in the context of clinical studies, reports on systematic validation of in vitro T cell assays are scarce so far. We recently validated our HLA-peptide multimer staining assay in a systematic manner so as to qualify the method for monitoring antigen-specific T cell responses after immunotherapy. METHODS: Parameters of the assay, specificity, precision, linearity, sensitivity, and robustness were assessed systematically. Experiments were designed to address specifically each parameter and are detailed. RESULTS: Nonspecific multimer staining was below the acceptance limit of 0.02% multimer(+) CD8(+) cells. The assay showed acceptable precision in all dimensions it was repeated (CV < 10%) and also demonstrated a linear detection (R2 > 0.99) of antigen specific cells. CONCLUSIONS: We succeeded in validating the HLA-multimer staining assay in a systematic manner. Additionally, we propose a technical framework and recommendations that can be applied for validating other T cell assessment methods. © 2016 International Clinical Cytometry Society.

Automated Analysis of Flow Cytometry Data to Reduce Inter-Lab Variation in the Detection of Major Histocompatibility Complex Multimer-Binding T Cells.

Manual analysis of flow cytometry data and subjective gate-border decisions taken by individuals continue to be a source of variation in the assessment of antigen-specific T cells when comparing data across laboratories, and also over time in individual labs. Therefore, strategies to provide automated analysis of major histocompatibility complex (MHC) multimer-binding T cells represent an attractive solution to decrease subjectivity and technical variation. The challenge of using an automated analysis approach is that MHC multimer-binding T cell populations are often rare and therefore difficult to detect. We used a highly heterogeneous dataset from a recent MHC multimer proficiency panel to assess if MHC multimer-binding CD8+ T cells could be analyzed with computational solutions currently available, and if such analyses would reduce the technical variation across different laboratories. We used three different methods, FLOw Clustering without K (FLOCK), Scalable Weighted Iterative Flow-clustering Technique (SWIFT), and ReFlow to analyze flow cytometry data files from 28 laboratories. Each laboratory screened for antigen-responsive T cell populations with frequency ranging from 0.01 to 1.5% of lymphocytes within samples from two donors. Experience from this analysis shows that all three programs can be used for the identification of high to intermediate frequency of MHC multimer-binding T cell populations, with results very similar to that of manual gating. For the less frequent populations (<0.1% of live, single lymphocytes), SWIFT outperformed the other tools. As used in this study, none of the algorithms offered a completely automated pipeline for identification of MHC multimer populations, as varying degrees of human interventions were needed to complete the analysis. In this study, we demonstrate the feasibility of using automated analysis pipelines for assessing and identifying even rare populations of antigen-responsive T cells and discuss the main properties, differences, and advantages of the different methods tested.

A Simple and Rapid Method for Quality Control of Major Histocompatibility Complex-Peptide Monomers by Flow Cytometry.

Major histocompatibility complex (MHC) multimers are essential tools in T cell immunomonitoring, which are employed both in basic and clinical research, as well as for assessing clinical samples during therapy. The generation of MHC monomers loaded with synthetic peptides is an elaborate and time-consuming process. It would be beneficial to assess the quality of these monomers prior to downstream applications. In this technical note, we describe a novel flow cytometry-based, cell-free, quick, and robust assay to check the quality of MHC monomers directly after refolding or after long-term storage.

Personalized peptide vaccine-induced immune response associated with long-term survival of a metastatic cholangiocarcinoma patient.

BACKGROUND & AIMS: We report a novel experimental immunotherapeutic approach in a patient with metastatic intrahepatic cholangiocarcinoma. In the 5year course of the disease, the initial tumor mass, two local recurrences and a lung metastasis were surgically removed. Lacking alternative treatment options, aiming at the induction of anti-tumor T cells responses, we initiated a personalized multi-peptide vaccination, based on in-depth analysis of tumor antigens (immunopeptidome) and sequencing. METHODS: Tumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands. RESULTS: Although several tumor-specific neo-epitopes were predicted in silico, none could be validated by mass spectrometry. Instead, a personalized multi-peptide vaccine containing non-mutated tumor-associated epitopes was designed and applied. Immunomonitoring showed vaccine-induced T cell responses to three out of seven peptides administered. The pulmonary metastasis resected after start of vaccination showed strong immune cell infiltration and perforin positivity, in contrast to the previous lesions. The patient remains clinically healthy, without any radiologically detectable tumors since March 2013 and the vaccination is continued. CONCLUSIONS: This remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma. LAY SUMMARY: Metastatic cholangiocarcinomas, cancers that originate from the liver bile ducts, have very limited treatment options and a fatal prognosis. We describe a novel therapeutic approach in such a patient using a personalized multi-peptide vaccine. This vaccine, developed based on the characterization of the patient's tumor, evoked detectable anti-tumor immune responses, associating with long-term tumor-free survival.

Concomitant Correction of a Soft-Tissue Fenestration with Keratinised Tissue Augmentation By Using A Rotated Double-Pedicle Flap During Second-Stage Implant Surgery- A Case Report.

Soft tissue deficiencies and defects around dental implants have been observed frequently. Soft-tissue defects after implant procedures originate from the process of modelling of periimplant mucosa and often cause aesthetic disharmony, food debris accumulation and soft tissue shrinkage. Periimplant mucogingival surgery focuses on creating an optimum band of keratinized tissue resulting in soft tissue architecture similar to the gingiva around natural teeth. A 23-year-old male reported to the Department of Periodontology with a complaint of gum soreness, foul smell and food accumulation at a site where a 3.75 x 11.5mm implant was placed previously. On clinical examination, fenestration of tissue above the cover screw was observed and there appeared to be a keratinized tissue of 1mm surrounding the implant. The case was managed by use of a rotated double-pedicle flap during second-stage implant surgery to correct the soft-tissue fenestration defect and to obtain a keratinized periimplant soft tissue. A periosteal bed was prepared by giving a horizontal incision at the mucogingival junction to a depth of 4 mm. Two split-thickness keratinized pedicles were dissected from the mesial and distal interproximal tissues near the implant. After rotation, both the pedicles were sutured to each other mid-buccally and the pedicles were rigidly immobilized with sutures. At 1 month, there was a 3mm band of stable and firm keratinized tissue over the underlying tissues. The procedure resulted in an aesthetic improvement due to enhanced soft tissue architecture and optimum integration between the peri-implant soft tissue and the final prosthesis.

The TGF-ß-inducible miR-23a cluster attenuates IFN-γ levels and antigen-specific cytotoxicity in human CD8⁺ T cells.

Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-ß are well established, we wondered whether TGF-ß could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-ß promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-ß up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-ß type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-ß, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-ß signaling.

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity.

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

A specific miRNA signature in the peripheral blood of glioblastoma patients.

The prognosis of patients afflicted by glioblastoma remains poor. Biomarkers for the disease would be desirable in order to allow for an early detection of tumor progression or to indicate rapidly growing tumor subtypes requiring more intensive therapy. In this study, we investigated whether a blood-derived specific miRNA fingerprint can be defined in patients with glioblastoma. To this end, miRNA profiles from the blood of 20 patients with glioblastoma and 20 age- and sex-matched healthy controls were compared. Of 1158 tested miRNAs, 52 were significantly deregulated, as assessed by unadjusted Student's t-test at an alpha level of 0.05. Of these, two candidates, miR-128 (up-regulated) and miR-342-3p (down-regulated), remained significant after correcting for multiple testing by Benjamini-Hochberg adjustment with a p-value of 0.025. The altered expression of these two biomarkers was confirmed in a second cohort of glioblastoma patients and healthy controls by real-time PCR and validated for patients who had received neither radio- nor chemotherapy and for patients who had their glioblastomas resected more than 6 months ago. Moreover, using machine learning, a comprehensive miRNA signature was obtained that allowed for the discrimination between blood samples of glioblastoma patients and healthy controls with an accuracy of 81% [95% confidence interval (CI) 78-84%], specificity of 79% (95% CI 75-83%) and sensitivity of 83% (95% CI 71-85%). In summary, our proof-of-concept study demonstrates that blood-derived glioblastoma-associated characteristic miRNA fingerprints may be suitable biomarkers and warrant further exploration.