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Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8+ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C+CD57+FcεRIγ-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C+CD57+FcεRIγlow-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C+CD8+ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

In this cross-sectional and longitudinal analysis of mapping the T-cell repertoire in kidney transplant recipients, we have investigated and validated T-cell clonality, immune repertoire chronology at rejection, and contemporaneous allograft biopsy quantitative tissue injury, to better understand the pathobiology of acute T-cell fraction, T-cell repertoire and antibody-mediated kidney transplant rejection. To follow the dynamic evolution of T-cell repertoire changes before and after engraftment and during biopsy-confirmed acute rejection, we sequenced 323 peripheral blood samples from 200 unique kidney transplant recipients, with (n=100) and without (n=100) biopsy-confirmed acute rejection. We report that patients who develop acute allograft rejection, have lower (p=0.01) T-cell fraction even before transplantation, followed by its rise after transplantation and at the time of acute rejection accompanied by high TCR repertoire turnover (p=0.004). Acute rejection episodes occurring after the first 6 months post-transplantation, and those with a component of antibody-mediated rejection, had the highest turnover; p=0.0016) of their T-cell repertoire. In conclusion, we validated that detecting repertoire changes in kidney transplantation correlates with post-transplant rejection episodes suggesting that T-cell receptor sequencing may provide recipient pre-transplant and post-transplant predictors of rejection risk.

Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensitivity and specificity. Plasma donor-derived cell-free DNA (dd-cfDNA) has been reported to accurately detect allograft rejection and injury in transplant recipients and shown to discriminate rejection from stable organ function in kidney transplant recipients. This study used a novel single nucleotide polymorphism (SNP)-based massively multiplexed PCR (mmPCR) methodology to measure dd-cfDNA in various types of renal transplant recipients for the detection of allograft rejection/injury without prior knowledge of donor genotypes. A total of 300 plasma samples (217 biopsy-matched: 38 with active rejection (AR), 72 borderline rejection (BL), 82 with stable allografts (STA), and 25 with other injury (OI)) were collected from 193 unique renal transplant patients; dd- cfDNA was processed by mmPCR targeting 13,392 SNPs. Median dd-cfDNA was significantly higher in samples with biopsy-proven AR (2.3%) versus BL (0.6%), OI (0.7%), and STA (0.4%) (p < 0.0001 all comparisons). The SNP-based dd-cfDNA assay discriminated active from non-rejection status with an area under the curve (AUC) of 0.87, 88.7% sensitivity (95% CI, 77.7⁻99.8%) and 72.6% specificity (95% CI, 65.4⁻79.8%) at a prespecified cutoff (>1% dd-cfDNA). Of 13 patients with AR findings at a routine protocol biopsy six-months post transplantation, 12 (92%) were detected positive by dd-cfDNA. This SNP-based dd-cfDNA assay detected allograft rejection with superior performance compared with the current standard of care. These data support the feasibility of using this assay to detect disease prior to renal failure and optimize patient management in the case of allograft injury.

Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensitivity and specificity. Plasma donor-derived cell-free DNA (dd-cfDNA) has been reported to accurately detect allograft rejection and injury in transplant recipients and shown to discriminate rejection from stable organ function in kidney transplant recipients. This study used a novel single nucleotide polymorphism (SNP)-based massively multiplexed PCR (mmPCR) methodology to measure dd-cfDNA in various types of renal transplant recipients for the detection of allograft rejection/injury without prior knowledge of donor genotypes. A total of 300 plasma samples (217 biopsy-matched: 38 with active rejection (AR), 72 borderline rejection (BL), 82 with stable allografts (STA), and 25 with other injury (OI)) were collected from 193 unique renal transplant patients; dd- cfDNA was processed by mmPCR targeting 13,392 SNPs. Median dd-cfDNA was significantly higher in samples with biopsy-proven AR (2.3%) versus BL (0.6%), OI (0.7%), and STA (0.4%) (p < 0.0001 all comparisons). The SNP-based dd-cfDNA assay discriminated active from non-rejection status with an area under the curve (AUC) of 0.87, 88.7% sensitivity (95% CI, 77.7⁻99.8%) and 72.6% specificity (95% CI, 65.4⁻79.8%) at a prespecified cutoff (>1% dd-cfDNA). Of 13 patients with AR findings at a routine protocol biopsy six-months post transplantation, 12 (92%) were detected positive by dd-cfDNA. This SNP-based dd-cfDNA assay detected allograft rejection with superior performance compared with the current standard of care. These data support the feasibility of using this assay to detect disease prior to renal failure and optimize patient management in the case of allograft injury.

Background Breast cancer-related lymphedema is the most common cause of lymphedema in the United States and occurs in up to 50% of individuals receiving axillary lymph node dissection (ALND). Lymphovenous bypass (LVB) at the time of ALND may prevent lymphedema, but long-term results and anastomotic patency are unclear. This study evaluates the feasibility and outcomes of performing immediate lymphatic reconstruction via coupler-assisted bypass (CAB). Methods This is a retrospective review of all patients undergoing prophylactic LVB following ALND at two tertiary care centers between 2018 and 2022. Patients were divided into cohorts based on whether they received the "standard" end-to-end (E-E) suturing or CAB technique. The primary outcome of interest was development of lymphedema. Quantitative and qualitative assessments for lymphedema were performed preoperatively and at 3, 6, 12, and 24 months postoperatively. Results Overall, 63 LVBs were performed, of which 24 lymphatics underwent immediate reconstruction via "CAB" and 39 lymphatics via "standard" end-to-end suture. Patient characteristics, including body mass index, and treatment characteristics, including radiation therapy, did not significantly differ between groups. CAB was associated with a greater mean number of lymphatics bypassed per vein (standard 1.7 vs. CAB 2.6, p = 0.0001) and bypass to larger veins (standard 1.2 vs. CAB 2.2 mm, p < 0.0001). At a median follow-up of 14.7 months, 9.1% (1/11) of individuals receiving CAB developed lymphedema. These rates were similar to those seen following standard bypass at 4.8% (1/21), although within a significantly shorter follow-up duration (standard 7.8 vs. CAB 14.7 months, p = 0.0170). Conclusion The CAB technique is a viable, effective technical alternative to the standard LVB technique. This comparative study of techniques in prophylactic LVB suggests that CABs maintain long-term patency, possibly due to the ease of anastomosing several lymphatics to single large caliber veins while reducing the technical demands of the procedure. [ABSTRACT FROM AUTHOR]

Hidradenitis suppurativa (HS), is a chronic inflammatory skin disorder that confers a substantial financial burden to patients. The aim of the current study was to assess the patient‐reported financial impact of HS. Patients presenting to a wound center between 2010 and 2021 were retrospectively reviewed. Demographics, comorbidities, and disease characteristics were collected. The financial impact of HS was assessed via the Comprehensive Score for Financial Toxicity ‐ Functional Assessment of Chronic Illness Therapy (COST‐FACIT) version 2 and investigator‐generated surveys. Of 199 patients contacted, 27.1% (n = 54) completed the survey. The majority were women (77.8%, n = 42) and had private health insurance (n = 30; 55.6%). Most patients (66.7%, n = 36) had Hurley stage III disease. Mean follow‐up was 2.3 + 2.8 years. The overall COST score was 19.7 + 12.4, indicating grade 1 financial toxicity (FT). Grade 0 FT was reported in 31.5% (n = 17) of patients, grade 1 in 37.0% (n = 20), grade 2 in 27.8% (n = 15), and grade 3 in 3.7% (n = 2). The mean self‐reported 12‐month out‐of‐pocket cost and credit scores were $2250 + 3269.24 and 674.6 + 95.3, respectively. Patients with private insurance had lower FT compared with Medicaid and Medicare (p = 0.003). Higher out‐of‐pocket costs were positively correlated with FT (p = 0.042), while higher credit scores were negatively correlated (p = 0.003). Patients with HS lesions in three or more anatomic regions reported the highest FT (p = 0.031). HS is a debilitating skin disorder that affects the livelihood of patients in a multifaceted manner. These patient‐reported outcomes highlight the impact that HS has on an individual's financial security, calling for further attention to this vulnerable population. [ABSTRACT FROM AUTHOR]

Background: The modified five-item frailty index (mFI-5) is a valuable tool for preoperative risk stratification of patients. There is a paucity of literature regarding the use of the mFI-5 in regard to males undergoing mastectomy. The aim of this study was to determine if the mFI-5 can predict negative postoperative outcomes. Methods: The 2005-2016 NSQIP database was queried to identify males aged 60 or older who underwent total mastectomy. Patient demographics, comorbidities, perioperative factors, and postoperative complications were collected. Patients were divided into cohorts based on whether their mFI-5 score was <2 or >2. Primary outcomes included prolonged postoperative length of stay (LOS), defined as greater than 75th percentile of the study population, and postoperative complications. Results: A total of 1,251 patients were identified, of whom 958 patients (76.6%) had mFI-5 scores <2 and 293 patients (23.4%) had mFI-5 scores >2. A significantly higher prevalence of medical complications occurred in the higher mFI group (6.1% vs. 1.6%, p<0.001). The mFI-5 was an independent risk factor for postoperative medical complications (OR 2.70, 95% CI, 1.28-5.68, p=0.009). Conclusion: This is the first study to evaluate the utility of mFI in predicting postoperative complications in males undergoing total mastectomies. We demonstrate that mFI has utility and is applicable as a risk stratification tool for males undergoing mastectomy. [ABSTRACT FROM AUTHOR]

Background: Up to 42% of all breast cancer patients undergo post-mastectomy reconstruction, however reconstructive techniques have not been widely studied in patients with triple negative breast cancer (TNBC). Reconstructive complications may delay adjuvant treatments; in TNBC, which inherently carries an increased risk of locoregional recurrence, this can greatly affect oncological outcomes. Therefore, we evaluate factors influencing choice of reconstructive techniques following mastectomy in TNBC patients and assess operative and oncologic safety outcomes., Methods: A single institution retrospective chart review identified TNBC patients who underwent post-mastectomy reconstruction between 2010 to 2020. Clinical characteristics collected included demographics, cancer history, reconstructive techniques [autologous-based reconstruction (ABR) vs . implant-based reconstruction (IBR)] and surgical and oncologic outcomes such as complications, recurrence, and mortality. Factors impacting whether patients underwent ABR versus IBR were assessed, as well as differences in outcomes between the two procedures. Statistical significance was defined as P<0.05., Results: During the 10-year period, 52.9% (n=127) of all post-mastectomy TNBC patients (n=240) underwent breast reconstruction, most frequently immediately after mastectomy (97.0%). Most patients underwent IBR compared to ABR (82.4% vs . 14.5%). Patients undergoing ABR were older than IBR patients (54.3 vs . 46.4 years; P=0.040) and had a higher body mass index (BMI; 30.0 vs . 26.1 kg/m 2 ; P=0.007). Patients more often pursued ABR if they had a prior breast cancer history (36.8% vs. 16.7%; P=0.041) or experienced TNBC recurrence (26.3% vs. 9.3%; P=0.034), while primary TNBC patients more often opted for IBR. Reconstructive type did not impact complications (ABR 31.6% vs. IBR 16.8%, P=0.131), recurrence (ABR 15.8% vs. IBR 13.0%, P=0.719), or mortality (ABR 0.0% vs. IBR 6.5%, P=0.593) rates., Conclusions: Factors such as age, BMI, and breast cancer history impacted choice of reconstructive technique among TNBC women. No differences in complications, recurrence, or mortality occur in these high-risk patients regardless of reconstructive technique, highlighting that neither ABR nor IBR is superior in regard to surgical and oncologic safety in post-mastectomy TNBC patients., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tbcr.amegroups.com/article/view/10.21037/tbcr-22-42/coif). The authors have no conflicts of interest to declare., (2023 Translational Breast Cancer Research. All rights reserved.)

In order to simplify a complex mixture of soluble proteins from tissues, a protocol to fractionate samples prior to two-dimensional (2D) gel electrophoresis has been developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins with DEAE-Sepharose can result in an increase in the number of detectable 2D gel spots. These gel spots are amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. The DEAE-Sepharose column fractionation acts to partition soluble proteins from cell extracts. Similarly, a SP-Sepharose column can fractionate soluble proteins and increase the number of detectable gel spots. Lastly, fractionation of cell extract with a phenyl Sepharose column can also result in an increase in the number of detectable 2D gel spots. This chapter describes an easy, inexpensive way to fractionate soluble proteins and a way to better profile proteomes.