The development of reproducible methods for deriving human embryonic stem cell (hESC) lines in compliance with good manufacturing practice (GMP) is essential for the development of hESC-based therapies. Although significant progress has been made toward the development of chemically defined conditions for the maintenance and differentiation of hESCs, efficient derivation of new hESCs requires the use of fibroblast feeder cells. However, GMP-grade feeder cell lines validated for hESC derivation are not readily available. We derived a fibroblast cell line (NclFed1A) from human foreskin in compliance with GMP standards. Consent was obtained to use the cells for the production of hESCs and to generate induced pluripotent stem cells (iPSCs). We compared the line with a variety of other cell lines for its ability to support derivation and self-renewal of hESCs. NclFed1A supports efficient rates (33%) of hESC colony formation after explantation of the inner cell mass (ICM) of human blastocysts. This compared favorably with two mouse embryonic fibroblast (MEF) cell lines. NclFed1A also compared favorably with commercially available foreskin fibroblasts and MEFs in promoting proliferation and pluripotency of a number of existing and widely used hESCs. The ability of NclFed1A to maintain self-renewal remained undiminished for up to 28 population doublings from the master cell bank. The human fibroblast line Ncl1Fed1A, produced in compliance with GMP standards and qualified for derivation and maintenance of hESCs, is a useful resource for the advancement of progress toward hESC-based therapies in regenerative medicine.
Abstract Background There are an estimated 1.3 million transgender adults in the US and about 25% undergo some form of gender-affirming surgery (GAS). Data on infectious complications following GAS is limited. We describe the epidemiology and incidence of infections following gender-affirming vaginoplasty (GAV). We also describe HIV screening and pre-exposure prophylaxis (PrEP) use in this population as transwomen are disproportionately affected by HIV and have the highest prevalence compared to any other group of US adults. Methods In this single-center retrospective cohort study, we identified 398 patients aged ≥18 years who underwent GAV at Oregon Health & Science University from 2016 to 2023. We reviewed medical records up to 6 months from the initial surgery and used standardized criteria to diagnose surgical site infections (SSI), urinary tract infections (UTI), and sexually transmitted infections (STI). Data around HIV screening and PrEP use was also obtained. Results 381 patients underwent primary vaginoplasty and 17 underwent revision of the primary surgery. 332 had standard penile inversion, 64 had robotic vaginoplasty, and 2 patients had intestinal vaginoplasty. UTI was the most common postoperative complication (incidence 13.8 per 100 individuals; n=55), followed by SSI (incidence 5.5 per 100 individuals; n=22). We identified 4 episodes of bacteremia related to surgery- all due to E coli- and one case of pelvic abscess. Only 2 STIs were identified and neither involved the neogenitalia- 1 was primary syphilis and the other was gonococcal urethritis. 16 patients had positive HIV serostatus. All 16 were prescribed ART and 11/16 had an undetectable viral load at the time of surgery. Only 22.5% of HIV seronegative patients had screening within a year before surgery and only 6% were on PrEP at the time of surgery. Conclusion In our study, UTIs and SSIs were the most common infections post-GAV. Serious infections were rare. We also identified a significant gap in HIV screening and PrEP uptake in this especially vulnerable group. Transgender surgery programs should incorporate HIV screening and counseling around PrEP. Disclosures Monica K. Sikka, MD, F2G: Grant/Research Support Christopher Evans, MD, ViiV: Grant/Research Support
Aim of the study: To develop a clinical grade fibrin gel for the culture of oral mucosal epithelial cells (OMEC) intended for ocular surface reconstruction in the treatment of limbal stem cell deficiency (LSCD).Materials and methods: Transparent fibrin gels composed of fibrinogen and thrombin were developed for the culture of epithelial cells. Oral mucosa was harvested from the buccal region of healthy volunteers and cultured as explants on fibrin gels. Tranexamic acid (TA), a clinically approved anti-fibrinolytic agent was added to prevent the fibrin gel from digesting due to cellular activity. The gels were stained for p63α (as a marker of poorly differentiated epithelial cells), CK19, CK13 and CK3 (expressed by OMEC). Epithelial cell stratification was observed using hematoxylin–eosin staining.Results: Addition of TA prevented gels from dissolving during the culture period. OMEC proliferated on the fibrin gel and attained confluence over a 2-week period (±2 d) and exhibited a typical epithelial, cobblestone morphology. Basal OMEC exhibited positive staining for p63α while the superficial cells exhibited positive staining for CK3. The cells expressed a strong immunoreactivity for CK19 and CK13 suggesting that they retained a normal oral epithelial phenotype.Conclusion: Fibrin gels, maintained in the presence of TA, to control the rate of substrate degradation, provide a more robust yet transparent substrate for the culture and transplantation of cultured OMEC. The fibrin gels are easily standardized, the components commercially available, and produced from clinically approved materials. The resulting stratified OMEC-derived epithelium displays characteristics similar to that of a human cornea, e.g. CK3 expression. The conventional dependence on a murine feeder layer for support of epithelial cells is unnecessary with this technique and hence, provides for an attractive alternative for treatment of LSCD.
Several new treatment modalities including fetal globin gene reactivation, allogeneic haematopoietic stem cell transplantation (alloHSCT) and gene therapy are being explored to correct the disparity in α/β globin chains in thalassaemia (de Dreuzy et al., 2016). Fetal haemoglobin (HbF) expression could decrease ineffective erythropoiesis by reducing the build-up and precipitation of α-globin chains (Soni, 2017). Thalidomide, a drug known for immunomodulating and anti-angiogenic properties, has been shown to induce γ-globin gene expression and increase the proliferation of erythroid cells (Gambari, 2010), which could be through the amplification of reactive oxygen species (ROS)-p38 mitogen-activated protein kinase (MAPK) signalling and histone H4 acetylation during adult erythropoiesis (Aerbajinai et al., 2007). Furthermore, hydroxyurea (HU) is known to increase haemoglobin (Hb) by HbF induction and reduction of inflammation and hypercoagulability (Keikhaei et al., 2015). A combination of thalidomide with HU for thalassaemia management has not been studied in a clinical setting to the best of our knowledge. In this single-centre, retrospective observational study, we reviewed the medical records of transplant ineligible [according to International Consensus Criteria (Angelucci et al., 2014): no fully matched sibling donor or very high-risk of transplant-related morbidity and mortality] patients of either sex with a confirmed diagnosis of β-thalassaemia major (severe anaemia, requirement of blood transfusions within the first two years of life) or β-thalassaemia intermedia (moderate anaemia, never transfused or sporadically transfused during infections or surgery, and transfusion dependent after 2 years of life) (Taher et al., 2009; Galanello & Origa, 2010), who were treated with thalidomide [dose: 50 mg once daily (OD) for <30 kg body weight (BW); 100 mg OD for >30 kg BW] and HU (500 mg daily) combination from Jul 2017 to Sep 2018 at Hemato-oncology Clinic, Vedanta Institute of Medical Sciences, Ahmedabad, India. This retrospective study was reviewed and approved by the ethics committee. Deferasirox, folic acid (5 mg) and aspirin (75/150 mg) were also administered. Medical charts had recorded informed consent for treatment and counselling regarding teratogenic potential of thalidomide. The study endpoints were: (i) increase in Hb levels from baseline at 1, 2 and 3 months; (ii) percentage of patients with major response (Hb increments >20 g/l) and minor response (Hb increments: 10–20 g/l) (Ren et al., 2018); and (iii) percentage of responders (transfusion independent after therapy) and non-responders (requiring transfusion after treatment) (Italia et al., 2009). The endpoints were also compared between thalassaemia intermedia and thalassaemia major subgroups. Of 25 patients in this study, 60% (n = 15) had β-thalassaemia intermedia and 40% (n = 10) had β-thalassaemia major. The majority (68%) were males, mean age was 14·4 years (range: 3–36 years), mean age at diagnosis was 31·4 months and at first transfusion, 28·3 months. Splenomegaly was observed in 80% of patients and splenectomy was performed in 20% of patients. Patients had varying degrees of iron overload (serum ferritin: 138–9777 ng/ml) with average Hb levels between 41 and 110 g/l at baseline. After thalidomide and HU combination treatment, Hb levels (g/l) significantly increased from baseline (76·4 ± 16·7) to 1 month (85·4 ± 17·3, P = 0·0106), 2 months (92·2 ± 19·4, P = 0·0002) and 3 months (94·6 ± 21·8 g/l, P = 0·0014), respectively; these changes were significant for the thalassaemia intermedia subgroup at all timepoints [baseline: 81·2 ± 16·7, 1 month: 94·6 ± 14·6 (P = 0·0107), 2 months: 102·6 ± 12·0 (P = 0·0004) and 3 months: 106·9 ± 11·9 (P = 0·0004)] but not significant for the thalassaemia major subgroup [baseline: 68·7 ± 16·0, 1 month: 72·0 ± 12·6 (P = 0·4352), 2 months: 78·6 ± 20·1 (P = 0·1387) and 3 months: 74·3 ± 20·7 (P = 0·4688)]. In the thalassaemia major subgroup, Hb levels were not available post-treatment in one patient at 1 month, and in two patients at 3 months, and in the thalassaemia intermedia subgroup in one patient each at 2 and 3 months (Table 1). Individual Hb values for patients with thalassaemia intermedia and thalassaemia major are presented in Figs 1A and 1B, respectively. Thalidomide was stopped in two patients in the thalassaemia major subgroup [one patient with Hb 105 g/l at 2 months underwent a bone marrow transplant (BMT), and another patient had a low Hb of 55 g/l after 3 months]. The overall response rates (major + minor response) were 47·8%, 58·3% and 68·2% at 1, 2 and 3 months, respectively (thalassaemia intermedia: 46·7%, 71·5% and 78·6%; thalassaemia major: 44·4%, 40% and 50%, respectively) (Fig 1C). In the thalassaemia intermedia subgroup (n = 15), two patients did not receive transfusion before and after treatment; one patient had increased Hb at all timepoints; the other had increased Hb at 1 month and was lost to follow-up. Of remaining 13 patients, 76·9% (10/13) were 'responders' and 23·1% (3/13) were 'non-responders'; in the three patients who required transfusion post-treatment, a decrease in transfusion frequency by 50% was seen in two whereas it remained same in the third patient. Transfusion details for two patients in the thalassaemia major (n = 10) group were not available post-treatment; of the remaining eight patients, 62·5% (5/8) were 'responders' whereas 37·5% (3/8) were 'non-responders' and continued blood transfusions with similar frequency. Overall, transfusion independence was reported in 71·4% (15/21) patients (Fig 1D). Total red and white blood cells, haematocrit and absolute neutrophil counts significantly increased while there was a non-significant decrease in serum ferritin levels. These changes were statistically significant for the thalassaemia intermedia subgroup but not for the thalassaemia major subgroup. A total of 15 adverse events were reported in 11 patients; of which thrombocytopenia was the most common adverse event reported (60%). No new safety concerns were observed. The study's limitations include its retrospective nature and a short follow-up duration of 3 months. Furthermore, the HbF levels were not measured in this study due to financial constraints of the patients; and hence, could not be reported. This is the largest such series of patients in which the combination of thalidomide and HU has been evaluated. We document maintenance of Hb levels, transfusion independence and reduction in iron overload and other haematological parameters in most of our patients. Our data suggests that thalidomide and HU combination therapy is effective in high-risk patients ineligible (no fully matched sibling donor/very high-risk of transplant-related morbidity and mortality) or not willing to undergo bone marrow transplant (poor disease characteristics/economic reasons) and could be used as a bridge to transplantation in ineligible thalassaemia patients who are awaiting identification of a donor or whose degree of iron overload predicts significant treatment-related toxicity. Prospective long-term efficacy and safety studies with thalidomide and HU combination may elicit better results to influence clinical practice. The authors wish to thank Mr. Shreekant Sharma (ISMPP CMPP™), Mr. Harshil Modi and Dr Venugopal Madhusudhana (ISMPP CMPP™) (Lambda Therapeutic Research Ltd.) for providing writing assistance, statistical analysis, and editorial assistance, respectively. The authors also thank Drs Deepak Bunger and Mujtaba Khan (Intas Pharmaceuticals Ltd.) for medical review inputs. The statistical analysis and manuscript development were supported by an unrestricted research grant by Intas Pharmaceuticals Limited. None declared. Sandip Shah had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors equally contributed to the concept and design, acquisition, analysis, or interpretation of data, and drafting/review of the manuscript.
Fusarium is a filamentous fungus that is ubiquitous in nature and can cause severe opportunistic infections in immunocompromised hosts. The association between Fusarium and hyper-IgE syndrome is exceedingly rare and has only been documented in a single report previously. A 44-year-old male, working as marijuana grower, with prior diagnosis of hyper-IgE syndrome and recurrent infections presented with enlarging right knee ulcer that did not respond to antimicrobial treatment. The patient was diagnosed with cutaneous fusariosis, confirmed with punch biopsy and positive wound cultures. The patient was managed with extended antifungal therapy (i.e., posaconazole) and surgical debridement resulting in remarkable improvement with wound healing leaving a pale scar. Fusarium should be considered in differential for cutaneous and invasive fungal infections in presence of cutaneous manifestations. Exposure to Cannabis plants is a noticeable risk factor. Multimodal approach involving systemic antifungals and wound debridement is essential for favorable outcome. Posaconazole was demonstrated to be a highly efficacious antifungal choice.
Abstract Background Hospital-acquired bloodstream infections (HABSI) are associated with increased mortality and decreased hospital quality metrics. This has led to an increased focus on blood culture stewardship. Little data exists regarding predictive factors of bacteremia in hospitalized patients. We aim to determine what clinical characteristics in patients were predictive of HABSI. Methods This is a retrospective case-control study of 540 patients with positive blood cultures admitted to our health system between September 1, 2017, to April 1, 2020. Electronic medical records of patients with positive blood cultures were independently reviewed to determine contamination versus true bacteremia. We looked at different clinical parameters and laboratory investigations within 24 hours of drawing blood cultures. Clinical variables were age ≥ 60 years, heart rate ≥ 90/minute, systolic blood pressure ≤ 90 mmHg or use of a vasopressor, oral temperature > 38°Celsius (100.4°Fahrenheit), white blood cells (WBC) count ≥12,000/µL, lymphocytes ≤ 1000/mm3, platelets < 150,000 /µL, and creatinine >2.0 mg/dL. Stepwise logistic regression analysis was used for predictive statistical model development. Results In a cohort of 481 patients with hospital-acquired bacteremia, 350 cases had true bacteremia and 131 cases were contaminated blood cultures. Stepwise regression analysis showed that white blood cell (WBC) count ≥ 12,000 cells/µL, lymphocyte count ≤ 1000/mm3, creatinine > 2.0 mg/dL, and oral temperature > 38°C (100.4°F) were associated with HABSI (R-square= 0.06, p value= 0.002). Conclusion Our findings suggest that WBC count, lymphocyte count, creatinine, and oral temperature together can be used to develop appropriate blood culture stewardship models in the inpatient setting. This may help minimize unnecessary blood cultures. Disclosures All Authors: No reported disclosures
Muir-Torre syndrome (MTS) is an autosomal dominant condition characterized by dermatological tumors along with visceral malignancies. The dermatological manifestations include recurrent sebaceous adenomas and keratoacanthomas. The commonly seen visceral malignancies are colorectal, gynecological, and urological. It is a variant of hereditary non-polyposis colorectal carcinoma syndrome (HNPCC). The underlying genetic mechanism is germline mutations in the DNA mismatch repair (MMR) genes leading to microsatellite instability (MSI), conferring an increased risk of developing malignancies. This is a case of a 57-year-old male patient with a history of colon cancer diagnosed at age 32 and multiple sebaceous adenomas. The patient also has a strong family history of cancer. They were referred to oncology after the immunohistochemical staining of a sebaceous adenoma showed loss of expression for MSH2 and MSH6. Next-generation sequencing identified a mutation in the MSH2 gene. These patients require genetic testing, counseling, and close follow-up with regular screening for cancer.
Pyoderma gangrenosum (PG) can be difficult to diagnose, leading to diagnostic delay which affects patient outcomes and increases health care utilization. Among different scenarios of diagnostic delay, atypical infections can mimic PG. Here, we present a case of extensive cutaneous leishmaniasis initially misdiagnosed as the superficial granulomatous variant of PG and describe diagnostic clues to aid in differentiation.
Department of Internal Medicine, Henry Ford Allegiance Health, Jackson, MI [email protected] The authors have no funding or conflicts of interest to disclose.
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