An immunogenetic basis for lung cancer risk.

Cancer risk is influenced by inherited mutations, DNA replication errors, and environmental factors. However, the influence of genetic variation in immunosurveillance on cancer risk is not well understood. Leveraging population-level data from the UK Biobank and FinnGen, we show that heterozygosity at the human leukocyte antigen (HLA)-II loci is associated with reduced lung cancer risk in smokers. Fine-mapping implicated amino acid heterozygosity in the HLA-II peptide binding groove in reduced lung cancer risk, and single-cell analyses showed that smoking drives enrichment of proinflammatory lung macrophages and HLA-II+ epithelial cells. In lung cancer, widespread loss of HLA-II heterozygosity (LOH) favored loss of alleles with larger neopeptide repertoires. Thus, our findings nominate genetic variation in immunosurveillance as a critical risk factor for lung cancer.

Isotope-Guided Metabolomics Reveals Divergent Incorporation of Valine into Different Flavor Precursor Classes in Chives.

Plants of the genus Allium such as chives, onions or garlic produce S-alk(en)yl cysteine sulfoxides as flavor precursors. Two major representatives are S-propenyl cysteine sulfoxide (isoalliin) and S-propyl cysteine sulfoxide (propiin), which only differ by a double bond in the C3 side chain. The propenyl group of isoalliin is derived from the amino acid valine, but the source of the propyl group of propiin remains unclear. Here, we present an untargeted metabolomics approach in seedlings of chives (Allium schoenoprasum) to track mass features containing sulfur and/or 13 C from labeling experiments with valine-13 C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13 C5 , but to a much lesser extent than isoalliin and related propenyl compounds. Our findings provide new insights into the biosynthetic pathways of flavor precursors in Allium species and open new avenues for future untargeted labeling experiments.

Clinical outcomes of ankle fractures in sub-Saharan Africa: a systematic review.

PURPOSE: Ankle fractures may cause disability and socioeconomic challenges, even when managed in a high-resource setting. The outcomes of ankle fractures in sub-Saharan Africa are not widely reported. We present a systematic review of the patient-reported outcomes and complications of patients treated for ankle fractures in sub-Saharan Africa. METHODS: Medline, Embase, Google Scholar and the Cochrane Central Register of Controlled Trials were searched, utilising MeSH headings and Boolean search strategies. Ten papers were included. Data included patient demographics, surgical and non-surgical management, patient-reported outcome measures and evidence of complications. RESULTS: A total of 555 patients with ankle fractures were included, 471 of whom were followed up (range 6 weeks-73 months). A heterogenous mix of low-quality observational studies and two methodologically poor-quality randomised trials demonstrated mixed outcomes. A preference for surgical management was found within the published studies with 87% of closed fractures being treated operatively. A total of five different outcome scoring systems were used. Most studies included in this review were published by well-resourced organisations and as such are not representative of the actual clinical practice taking place. CONCLUSION: The literature surrounding the clinical outcomes of ankle fractures in sub-Saharan Africa is sparse. There appears to be a preference for surgical fixation in the published literature and considering the limitations in surgical resources across sub-Saharan Africa this may not be representative of real-life care in the region.

The effect of human immunodeficiency virus and human papillomavirus strain diversity on the progression of anal squamous intraepithelial lesions.

Anal squamous cell carcinoma (SCC) is a human papillomavirus (HPV)-mediated malignancy with increasing incidence. Human immunodeficiency virus (HIV) infection is a significant risk factor for anal SCC; however, it is unknown if HIV infection alters anal lesion progression and HPV strain profile. This study aims to determine whether HIV coinfection is associated with progression of HPV-mediated anal lesions and on their HPV strain diversity. This is a retrospective cohort study of adults with anal squamous intraepithelial lesion (SIL) who presented for anorectal sampling between 2010 and 2019. Using the full cohort, we performed clinicopathologic epidemiologic analysis of HIV coinfection on lesion progression. Using a subset of patients, we conducted molecular analysis of HPV strain diversity as related to HIV status and progression. Our cohort included 2203 individuals, of which 940 (43%) were HIV+. HIV+ status was associated with faster progression at all levels of dysplasia. Our molecular cohort included 329 adults, of which 190 (57.8%) were HIV+. HIV+ status was associated with higher HPV strain diversity (median: 7 [5-9] versus median: 4 [4-6], P < .001). Latent class analysis identified specific HPV strain signatures associated with progression. We demonstrate that HIV+ individuals had faster rates of anal SIL progression and that almost all HPV strains were more prevalent in anal samples from HIV+ adults. Our results imply that HIV+ adults with anal SIL should undergo more frequent screening and obtain HPV genotyping at initial presentation, as it shows value as a biomarker of lesion progression.

The Possible Pathogenic Role of IgG4-Producing Plasmablasts in Stricturing Crohn's Disease.

BACKGROUND: Crohn's disease (CD) is a condition on the spectrum of inflammatory bowel disease that affects up to 20 people per 100,000 in the US annually, and with incidence increasing. One of the most significant sources of morbidity in CD is the formation of strictures, with resultant intestinal blockage a common indication for hospitalization and surgical intervention in these patients. The pathophysiology of stricture formation is not fully understood. However, the fibroplasia that leads to fibrostenotic stricture formation may have shared pathophysiology with IgG4-related fibrosis. SUMMARY: Initial intestinal inflammation recruits innate immune cells, such as neutrophils, that secrete IL-1ß and IL-23, which induces a type 17 CD4+ T-helper T-cell (Th17)-mediated adaptive immune response. These CD4+ Th17 T cells also contribute to inflammation by secreting proinflammatory cytokines such as IL-17 and IL-21. IL-21 recruits and stimulates CD4+ T follicular helper (Tfh) cells, which secrete more IL-21. This causes ectopic germinal center formation, recruiting and stimulating naïve B cells. The IL-17 and IL-21 produced by Th17 cells and Tfh cells also induce IgG4 plasmablast differentiation. Finally, these IgG4-producing plasmablasts secrete platelet-derived growth factor (PDGF), which activates local PDGF-receptor expressing fibroblasts and myofibroblasts, resulting in uncontrolled fibroplasia.

Human Immunodeficiency Virus Infection Promotes Human Papillomavirus-Mediated Anal Squamous Carcinogenesis: An Immunologic and Pathobiologic Review.

BACKGROUND: Anal squamous cell carcinoma (SCC) is a rare gastrointestinal malignancy with rising incidence, both in the United States and internationally. The primary risk factor for anal SCC is human papillomavirus (HPV) infection. However, there is a growing burden of disease in patients with human immunodeficiency virus (HIV) and HPV coinfection, with the incidence of anal SCC significantly increasing in this population. This is particularly true in HIV-infected men. The epidemiologic correlation between HIV-HPV coinfection and anal SCC is established; however, the immunologic mechanisms underlying this relationship are not well understood. SUMMARY: HIV-related immunosuppression due to low circulating CD4+ T cells is one component of increased risk, but other mechanisms, such as the effect of HIV on CD8+ T lymphocyte tumor infiltration and the PD-1/PD-L1 axis in antitumor and antiviral response, is emerging as significant contributors. The goal of this article is to review existing research on HIV-HPV coinfected anal SCC and precancerous lesions, propose explanations for the detrimental synergy of HIV and HPV on the pathogenesis and immunologic response to HPV-associated cancers, and discuss implications for future treatments and immunotherapies in HIV-positive patients with HPV-mediated anal SCC. Key Messages: The incidence of anal squamous cell carcinoma is increased in human immunodeficiency virus (HIV)-infected patients, even in patients on highly active antiretroviral therapy. Locoregional HIV infection may enhance human papillomavirus oncogenicity. Chronic inflammation due to HIV infection may contribute to CD8+ T lymphocyte exhaustion by upregulating PD-1 expression, thereby blunting cytotoxic antitumor response.

Total knee replacement in a transtibial amputee.

We present the case of a man in his 60s with a transtibial amputation (TTA) undergoing total knee replacement (TKR) for symptomatic osteoarthritis (OA). It is unusual to develop OA in the ipsilateral knee to TTA; and while it is postulated that this is because patients preferentially load their unaffected limb to protect the TTA-sided knee, there is also the ability to offload specific knee compartments through prosthetic adjustment. When planning TKR in such patients, it is important to consider several technical challenges in order to prevent a poor outcome. The literature is sparse with evidence to guide decision-making, and this case report and literature review aims to summarise our preoperative planning and intraoperative technique, which ultimately resulted in a good outcome.

Retrosacral Morel-Lavallée lesion: resolution with ultrasound-guided drainage and sclerotherapy.

Morel-Lavallée lesions are chronic seromas due to closed degloving injuries, resulting from blunt trauma. They most commonly occur over the greater trochanteric, gluteal and flank regions. We present a case of retrosacral Morel-Lavallée lesion. Initial ultrasound demonstrated a fluid collection lying between the subcutaneous fat and the underlying fascia superficial to the sacrum. Following repeated ultrasound-guided aspirations, further recurrence of a superficial pre-sacral seroma was confirmed with MRI. Ultrasound-guided aspiration was performed and 100 mg of injectable doxycycline was instilled into the lesion. 4 months after sclerotherapy, the patient was asymptomatic, and follow-up MRI demonstrated no residual fluid collection or complication. This case demonstrates the value of using MRI in conjunction with ultrasound to characterize Morel-Lavallée lesions in an atypical site and in confirming response to treatment, in addition to the use of sclerotherapy for treatment of a lesion refractory to repeated aspiration.

Novel technique of explantation of rigid phakic iris-claw lens and cataract extraction by sutureless manual small-incision surgery.

A 38-year-old patient presented to us with complaints of blurred vision and photophobia in the left eye with an uncorrected visual acuity of 20/400, improving to 20/60 with pinhole. The patient underwent phakic iris-claw lens surgery 15 years ago for high myopia. On examination, there was anterior chamber rigid phakic iris-claw lens along with complicated cataract. We planned for sutureless self-sealing 6.5 mm sclerocorneal tunnel for explantation of rigid phakic iris-claw lens along with cataract extraction with irrigating vectis. There was postoperative reduction in astigmatism due to incision planned on steep axis, and visual acuity improved to 20/30 uncorrected. This technique provides significant advantages from the previously described techniques in terms of decreased postop astigmatism, no need for sutures, no issues of chamber instability and iris trauma and without the need for phacoemulsification.

Persistently poor vision in dim illumination after implantation of XtraFocus small-aperture IOL (Morcher).

A 21-year-old patient presents to us with complaints of blurred vision and photophobia in the left eye, with an uncorrected visual acuity of 20/100 improving to 20/30 with pinhole and diagnostic rigid gas permeable lens trial. He had a history of trauma with subsequent cataract extraction, with residual irregular astigmatism and traumatic mydriasis. XtraFocus Pinhole intraocular lens (Morcher) was implanted in the left eye. One week postoperatively, the left eye uncorrected visual acuity improved to 20/30, uncorrected intermediate visual acuity improved to 20/40, and uncorrected near visual acuity improved to J4. The glare and photophobia resolved completely. Surprisingly, the patient complained of severely poor vision in dim illumination. His vision was limited to bare perception of objects and hand movements close to the face. He started facing difficulties in major activities such as driving at night and in dark ambient surroundings such as movie theatres, which persisted to the extent of necessitating explantation of the implant.

Persistent troublesome floaters necessitating the explantation of XtraFocus Pinhole IOL (Morcher).

A 41-year-old patient presented with blurred vision and photophobia in the left eye with an uncorrected visual acuity of 20/150, improving to 20/30 with pinhole and diagnostic rigid gas permeable lens trial. He had a history of trauma with subsequent cataract extraction with residual irregular astigmatism and traumatic mydriasis. XtraFocus Pinhole intraocular lens (Morcher) was implanted in the left eye and the vision improved to 20/40. Postoperatively, the patient experienced significant floaters which persisted to the extent of necessitating explantation of implant.

Xtra focus pinhole IOL (Morchers GMBH) a novel approach to tackle irregular astigmatism and large pupillary defects with a single step surgery.

A 69-year-old patient presented to us with traumatic mydriasis with irregular pupil measuring 7 mm, with superior loss of iris tissue and large inferior peripheral iridotomy and pseudophakia. The patient had history of blunt trauma 3 years ago in a fire cracker injury. He was operated elsewhere primarily after the trauma for cataract surgery with intraocular lens implantation and had suboptimal visual outcome with glare and photophobia. He presented to us with irregular pupil and inferior iridectomy with pseudophakia. The uncorrected visual acuity was 20/150 improving to 20/50 with glasses. He had a history of cataract surgery with intraocular lens (IOL) implantation done elsewhere several years back. The patient was not a diabetic or hypertensive. There was a para central corneal scar causing irregular corneal astigmatism. Extra focus pinhole IOL was implanted in sulcus having a pinhole aperture 1.36 mm. Preoperative total corneal higher-order aberrations were 3.3 µ and total corneal coma was 0.97 µ. Postoperatively uncorrected distance visual acuity improved to 20/40 intermediate uncorrected visual acuity improved to 20/30 and uncorrected near visual acuity was J3.

Importance of clinical signs leading to the diagnosis in a case of microbiological smear negative ocular microsporidiosis.

A 28-year-old patient presented to us with multifocal coarse raised epithelial lesions in the left eye associated with pain watering redness and blurred vision with a visual acuity of 20/40 in the left eye. The patient had been managed elsewhere with a course of topical moxifloxacin eye-drops four times a day and topical steroids (prednisolone acetate) 1% three times a day for 2 weeks without any resolution, which was stopped 2 days ago prior to presentation at our centre. Gram stain was negative for bacteria as well as microsporidial spores. 10% KOH was negative for fungal hyphae. Based on strong clinical signs of corneal microsporidiosis, in spite of the negative microbiology smear, the patient was started on voriconazole eye-drops five times a day. The lesions started resolving in 5 days and completely healed after 17 days of therapy. No relevant history pertaining to exposure of contaminated water, swimming or history of trauma could be elicited.

Tracheal Replacement Therapy with a Stem Cell-Seeded Graft: Lessons from Compassionate Use Application of a GMP-Compliant Tissue-Engineered Medicine.

Tracheal replacement for the treatment of end-stage airway disease remains an elusive goal. The use of tissue-engineered tracheae in compassionate use cases suggests that such an approach is a viable option. Here, a stem cell-seeded, decellularized tissue-engineered tracheal graft was used on a compassionate basis for a girl with critical tracheal stenosis after conventional reconstructive techniques failed. The graft represents the first cell-seeded tracheal graft manufactured to full good manufacturing practice (GMP) standards. We report important preclinical and clinical data from the case, which ended in the death of the recipient. Early results were encouraging, but an acute event, hypothesized to be an intrathoracic bleed, caused sudden airway obstruction 3 weeks post-transplantation, resulting in her death. We detail the clinical events and identify areas of priority to improve future grafts. In particular, we advocate the use of stents during the first few months post-implantation. The negative outcome of this case highlights the inherent difficulties in clinical translation where preclinical in vivo models cannot replicate complex clinical scenarios that are encountered. The practical difficulties in delivering GMP grafts underscore the need to refine protocols for phase I clinical trials. Stem Cells Translational Medicine 2017;6:1458-1464.

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds.

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3-8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.

Two-Stage Priming of Allogeneic Natural Killer Cells for the Treatment of Patients with Acute Myeloid Leukemia: A Phase I Trial.

UNLABELLED: Human Natural Killer (NK) cells require at least two signals to trigger tumor cell lysis. Absence of ligands providing either signal 1 or 2 provides NK resistance. We manufactured a lysate of a tumour cell line which provides signal 1 to resting NK cells without signal 2. The tumor-primed NK cells (TpNK) lyse NK resistant Acute Myeloid Leukemia (AML) blasts expressing signal 2 ligands. We conducted a clinical trial to determine the toxicity of TpNK cell infusions from haploidentical donors. 15 patients with high risk AML were screened, 13 enrolled and 7 patients treated. The remaining 6 either failed to respond to re-induction chemotherapy or the donor refused to undergo peripheral blood apheresis. The conditioning consisted of fludarabine and total body irradiation. This was the first UK trial of a cell therapy regulated as a medicine. The complexity of Good Clinical Practice compliance was underestimated and led to failures requiring retrospective independent data review. The lessons learned are an important aspect of this report. There was no evidence of infusional toxicity. Profound myelosuppression was seen in the majority (median neutrophil recovery day 55). At six months follow-up, three patients treated in Complete Remission (CR) remained in remission, one patient infused in Partial Remission had achieved CR1, two had relapsed and one had died. One year post-treatment one patient remained in CR. Four patients remained in CR after treatment for longer than their most recent previous CR. During the 2 year follow-up six of seven patients died; median overall survival was 400 days post infusion (range 141­910). This is the first clinical trial of an NK therapy in the absence of IL-2 or other cytokine support. The HLA-mismatched NK cells survived and expanded in vivo without on-going host immunosuppression and appeared to exert an anti-leukemia effect in 4/7 patients treated. TRIAL REGISTRATION: ISRCTN trial registry ISRCTN11950134.

Assessment of the effector function of CMV-specific CTLs isolated using MHC-multimers from granulocyte-colony stimulating factor mobilized peripheral blood.

BACKGROUND: Adoptive transfer of CMV-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in immunocompromised patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or months after G-CSF mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. Despite the tolerogenic effects associated with G-CSF mobilization, recent studies described that CMV-primed T cells generated from mobilized donors remain functional. METHODS: MHC-multimers are potent tools that allow the rapid production of antigen-specific CTLs. Therefore, in the present study we have assessed the feasibility and efficacy of CMV-specific CTL manufacture from G-CSF mobilized apheresis using MHC-multimers. RESULTS: CMV-specific CTLs can be efficiently isolated from G-CSF mobilized samples with Streptamers and are able to express activation markers and produce cytokines in response to antigenic stimulation. However, this anti-viral functionality is moderately reduced when compared to non-mobilized products. CONCLUSIONS: The translation of Streptamer technology for the isolation of anti-viral CTLs from G-CSF mobilized PBMCs into clinical practice would widen the number of patients that could benefit from this therapeutic strategy, although our results need to be taken into consideration before the infusion of antigen-specific T cells obtained from G-CSF mobilized samples.

CMV-specific T cell isolation from G-CSF mobilized peripheral blood: depletion of myeloid progenitors eliminates non-specific binding of MHC-multimers.

BACKGROUND: Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product. METHODS: In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples. RESULTS: After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved. CONCLUSIONS: G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired alloreactive cell infusion.

Manufacturing of highly functional and specific T cells for adoptive immunotherapy against virus from granulocyte colony-stimulating factor-mobilized donors.

BACKGROUND AIMS: Cytomegalovirus (CMV) reactivation remains an important risk after hematopoietic stem cell transplantation, which can be effectively controlled through adoptive transfer of donor-derived CMV-specific T cells (CMV-T). CMV-T are usually obtained from donor peripheral blood mononuclear cells (PBMCs) collected before G-CSF mobilization. Despite previous studies that showed impaired T-cell function after granulocyte colony-stimulating factor (G-CSF) mobilization, recent publications suggest that G-CSF-primed PBMCs retain anti-viral function and are a suitable starting material for CMV-T manufacturing. The objective of this study was to assess the feasibility of generating CMV-T from G-CSF-mobilized donors by use of the activation marker CD137 in comparison with conventional non-primed PBMCs. METHODS: CMV-T were isolated from G-CSF-mobilized and non-mobilized donor PBMCs on the basis of CMVpp65 activation-induced CD137 expression and expanded during 3 weeks. Functional assays were performed to assess antigen-specific activation, cytokine release, cytotoxic activity and proliferation after anti-genic re-stimulation. RESULTS: We successfully manufactured highly specific, functional and cytotoxic CMV-T from G-CSF-mobilized donor PBMCs. Their anti-viral function was equivalent to non-mobilized CMV-T, and memory phenotype would suggest their long-term maintenance after adoptive transfer. CONCLUSIONS: We confirm that the use of an aliquot from G-CSF-mobilized donor samples is suitable for the manufacturing of CMV cellular therapies and thereby abrogates the need for successive donations and ensures the availability for patients with unrelated donors.