Outcomes of Calvarial and Soft-Tissue Reconstruction with Latissimus Dorsi-Rib Osteomyocutaneous Free Flap.

BACKGROUND: The latissimus dorsi-rib osteomyocutaneous free flap (LDRF) has been used for autologous reconstruction of large composite calvarial and scalp defects. In this study, the authors aim to present clinical and patient-reported outcomes after LDRF reconstruction. METHODS: An anatomical study was conducted to evaluate the distribution of the connecting perforators between the thoracodorsal and intercostal systems. An institutional review board-approved retrospective review of 10 patients who underwent LDRF with one or two ribs for treatment of cranial defects was conducted. Patient-reported outcomes regarding quality of life, neurologic status, and functional status were evaluated using validated surveys. One-way analysis of variance and post hoc Tukey tests were used for anatomical outcomes. Preoperative and postoperative scores were compared using paired t tests. RESULTS: The tenth rib (4.65 ± 2.01) followed by the ninth rib (3.7 ± 1.63) had the highest number of perforators. A combination of the ninth and eleventh ribs exhibited maximal perforator number and pedicle length. All patients had stable LDRF reconstructions. Eight patients completed both preoperative and postoperative questionnaires; Median clinical follow-up was 48 months (range, 34 to 70 months). Scores trended toward improvement but did not reach statistical significance on the Karnofsky Performance Scale ( P = 0.22), the Functional Independence Measure (Motor, P = 0.52; Cognitive, P = 0.55), or the Headache Disability Index ( P = 0.38). The minimum clinically important difference was surpassed, demonstrating improvement of function for 71% of patients on the Barthel Index and 63% on the Selective Functional Movement Assessment test. CONCLUSION: The LDRF can improve cognitive and physical functional status in complex patients with prior failed reconstructions for composite scalp and skull defects. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

A Systematic Review of the Outcomes of Carpal Ligament Release in Severe Carpal Tunnel Syndrome.

PURPOSE: Indications for surgical treatment of severe carpal tunnel syndrome (CTS) are controversial. The aim of this study was to review the outcomes reported in the literature of carpal tunnel release in patients with severe CTS. METHODS: A systematic review of the outcomes of carpal tunnel release in patients with severe CTS was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Outcome measures included change in symptoms, sensation (2-point discrimination, light touch), thenar atrophy, strength (power and pinch grip), electrophysiology, median nerve morphology, and patient-reported outcome measures. Outcomes are reported by ranges of the percentage of patients/hands improved in the included studies. RESULTS: Thirty-eight papers were selected, representing 2,531 patients and 2,712 hands. Demographic information on age and sex were available for a total of 1,542 patients. Mean age ranged from 49.8 to 83 years and 72% were female. All studies that assessed patient-reported outcome measures before and after surgery reported significant improvements. Complete resolution of paresthesia occurred in 55%-98% of hands across different studies. Resolution of numbness occurred in between 39% and 94% of hands. Pain completely resolved in 64%-100% and weakness in 60%-75% of hands. Two-point discrimination and light touch improved postoperatively. Power grip, key, tripod, index-thumb pulp pinch, and thumb opposition increased. Motor and sensory amplitudes, distal motor latencies, and sensory conduction velocities improved. Patient-reported outcomes indicated symptomatic improvement and reduced disability. CONCLUSIONS: Symptomatic improvement following carpal tunnel release in patients with severe CTS can occur. Patients should be counseled about the unpredictability of the outcomes and factors that might affect outcomes. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Ex Vivo Normothermic Perfusion of Human Upper Limbs.

BACKGROUND: Ischemia-reperfusion injury remains a primary concern in upper extremity transplantation. Ex vivo normothermic perfusion (EVNP) enables near-physiological organ preservation, avoiding the deleterious effects of hypoxia and cooling. We investigated the effectiveness of human limb EVNP compared with static cold storage (SCS). METHODS: Twenty human upper extremities were procured. Ten were perfused at 38 °C with an oxygenated red blood cell-based solution, and contralateral limbs served as SCS control (4 °C). EVNP was terminated with systolic arterial pressure ≥115 mm Hg, compartment fullness, or a 20% decline in oxygen saturation. Weight, contractility, compartment pressure, tissue oxygen saturation, and uptake rates were assessed. Perfusate fluid dynamics, gases, electrolytes, and metabolites were measured. Myocyte injury scores and liquid chromatography-mass spectrometry analysis were performed. RESULTS: EVNP duration was 41.6 ± 9.4 h. Vascular resistance averaged 173.0 ± 29.4 mm Hg × min/L. Weight change and compartment pressures were 0.4 ± 12.2% ( P = 0.21) and 21.7 ± 15.58 mm Hg ( P = 0.003), respectively. Arterial and venous carbon dioxide partial pressure, oxygen saturation, and pH were 509.5 ± 91.4 mm Hg, 15.7 ± 30.2 mm Hg, 87.4 ± 11.4%, and 7.3 ± 0.2, respectively. Oxygen uptake rates averaged 5.7 ± 2.8 mL/min/g. Lactate reached 20 mmol/L after 15 (interquartile range = 6) h. Limb contractility was preserved for 30.5 (interquartile range = 15.8) h ( P < 0.001) and negatively correlated with perfusate potassium (ρ = -0.7, P < 0.001). Endpoint myocyte injury scores were 28.9 ± 11.5% (EVNP) and 90.2 ± 11.8% (SCS) ( P < 0.001). A significant increase in taurine ( P = 0.002) and decrease in tryptophan ( P = 0.002) were detected. Infrared thermography and indocyanine green angiography confirmed the presence of peripheral perfusion. CONCLUSIONS: EVNP can overcome the limitations of cold preservation by extending preservation times, enabling limb quality assessment, and allowing limb reconditioning before transplantation.

Histomorphometry in Peripheral Nerve Regeneration: Comparison of Different Axon Counting Methods.

BACKGROUND: Histomorphometry quantitatively evaluates nerve regeneration. Total myelinated fiber count (TMFC) is most accurately obtained manually across full nerve cross-sections, but most researchers opt for automated, sampled analysis. Few of the numerous techniques available have been validated. The goal of this study was to compare common histomorphometric methods (full manual [FM], sampled manual [SM], and sampled automatic [SA]) to determine their reliability and consistency. MATERIAL AND METHODS: Twenty-four rats underwent sciatic nerve (SN) repair with 20mm isografts; SNs distal to the graft were analyzed. TMFC was manually determined in each full cross-section. Counts were also extrapolated from sampled fields, both manually and automatically with ImageJ software. Myelinated fiber diameter, axon diameter, and myelin sheath thickness were measured manually in full and sampled fields; G-ratio was calculated. Repeated-measures MANOVA, Spearman correlation, and Wilcoxon signed-rank tests were performed. A systematic review of histomorphometry in rat SN repair was performed to analyze the variability of techniques in the literature. RESULTS: FM TMFC was 13,506 ± 4,217. Both sampled methods yielded significantly different TMFCs (SM:14.4 ± 13.4%, P< 0.001; SA:21.8 ± 44.7%, P = 0.037). All three methods strongly correlated with each other, especially FM and SM (rs = 0.912, P< 0.001). FM fiber diameter, axon diameter, and myelin sheath thickness did not differ from SM (P = 0.493, 0.209, and 0.331, respectively). 65% of papers used sampling; 78% utilized automated or semi-automated analysis. Software, sampling, and histomorphometric parameters varied widely. CONCLUSION: SM and SA analysis are reliable with standardized, systematic sampling. Transparency is essential to allow comparison of data; meanwhile, researchers must be cognizant of the wide variety of methodologies in the literature.

Leukoreduction in ex vivo perfusion circuits: comparison of leukocyte depletion efficiency with leukocyte filters.

BACKGROUND: Leukodepletion of whole blood-based perfusates remains a challenge in experimental models of ex vivo perfusion. This study investigated the leukoreduction efficacy of the commonly used LeukoGuard LG Arterial and BC2 Cardioplegia filters. METHODS: Eleven liters of washed porcine blood was used to evaluate the filtration efficiency of LG (n = 6) and BC2 (n = 5) filters. Filter efficacy was tested by passing 1 L of washed blood through each filter. Complete blood count was performed to detect a reduction of white blood cells, red blood cells, and hemoglobin concentration. RESULTS: The BC2 Cardioplegia filter showed a significant reduction in white blood cell count (13.16 ± 4.2 × 103 cells/µL pre-filtration, 0.62 ± 0.61 cells/µL post-filtration, p = 0.005), red blood cell count (9.18 ± 0.16 × 106 cells/µL pre-filtration, 9.02 ± 0.16 × 106 cells/µL post-filtration, p = 0.012) and hemoglobin concentration (15.89 ± 0.66 g/dL pre-filtration, 15.67 ± 0.83 g/dL post-filtration, p = 0.017). Platelet reduction in the LG filter group was statistically significant (13.23 ± 13.98 × 103 cells/µL pre-filtration, 7.15 ± 3.31 × 103 cells/µL post-filtration, p = 0.029), but no difference was seen in the BC2 group. There was no significant difference in white blood cell count in the LG filter group (10.12 ± 3.0 × 103 cells/µL pre-filtration, 10.32 ± 2.44 × 103 cells/µL post-filtration, p = 0.861). CONCLUSION: Our results suggest that the LG filter should not be used in ex vivo perfusion circuits for the purpose of leukodepletion. The BC2 filter can be used in EVP circuits with flow rates of less than 350 mL/min. Alternatively, perfusate may be leukodepleted before perfusion.

Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer.

Data from mouse tumor models suggest that tumor-associated monocyte/macrophage lineage cells (MMLCs) dampen antitumor immune responses. However, given the fundamental differences between mice and humans in tumor evolution, genetic heterogeneity, and immunity, the function of MMLCs might be different in human tumors, especially during early stages of disease. Here, we studied MMLCs in early-stage human lung tumors and found that they consist of a mixture of classical tissue monocytes and tumor-associated macrophages (TAMs). The TAMs coexpressed M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors. Functionally, TAMs did not primarily suppress tumor-specific effector T cell responses, whereas tumor monocytes tended to be more T cell inhibitory. TAMs expressing relevant MHC class I/tumor peptide complexes were able to activate cognate effector T cells. Mechanistically, programmed death-ligand 1 (PD-L1) expressed on bystander TAMs, as opposed to PD-L1 expressed on tumor cells, did not inhibit interactions between tumor-specific T cells and tumor targets. TAM-derived PD-L1 exerted a regulatory role only during the interaction of TAMs presenting relevant peptides with cognate effector T cells and thus may limit excessive activation of T cells and protect TAMs from killing by these T cells. These results suggest that the function of TAMs as primarily immunosuppressive cells might not fully apply to early-stage human lung cancer and might explain why some patients with strong PD-L1 positivity fail to respond to PD-L1 therapy.

Human lung tumor FOXP3+ Tregs upregulate four "Treg-locking" transcription factors.

Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses, but the properties of human intratumoral Tregs are still largely unknown, in part due to significant methodologic problems. We studied the phenotypic, functional, epigenetic, and transcriptional features of Tregs in 92 patients with non-small-cell lung cancer, comparing the features of Tregs within tumors versus corresponding blood, lung, and lymph node samples. Intratumoral Treg numbers and suppressive function were significantly increased compared with all other sites but did not display a distinctive phenotype by flow cytometry. However, by undertaking simultaneous evaluation of mRNA and protein expression at the single-cell level, we demonstrated that tumor Tregs have a phenotype characterized by upregulated expression of FOXP3 mRNA and protein as well as significantly increased expression of EOS, IRF4, SATB1, and GATA1 transcription factor mRNAs. Expression of these "Treg-locking" transcription factors was positively correlated with levels of FOXP3 mRNA, with highest correlations for EOS and SATB1. EOS had an additional, FOXP3 mRNA-independent, positive correlation with FOXP3 protein in tumor Tregs. Our study identifies distinctive features of intratumoral Tregs and suggests that targeting Treg-locking transcription factors, especially EOS, may be of clinical importance for antitumor Treg-based therapy.

Radium-228 analysis of natural waters by Cherenkov counting of Actinium-228.

The activities of (228)Ra in natural waters were determined by the Cherenkov counting of the daughter nuclide (228)Ac. The radium was pre-concentrated on MnO(2) and the radium purified via ion exchange and, after a 2-day period of incubation to allow for secular equilibrium between the parent-daughter (228)Ra((228)Ac), the daughter nuclide (228)Ac was isolated by ion exchange according to the method of Nour et al. [2004. Radium-228 determination of natural waters via concentration on manganese dioxide and separation using Diphonix ion exchange resin. Appl. Radiat. Isot. 61, 1173-1178]. The Cherenkov photons produced by (228)Ac were counted directly without the addition of any scintillation reagents. The optimum Cherenkov counting window, sample volume, and vial type were determined experimentally to achieve optimum Cherenkov photon detection efficiency and lowest background count rates. An optimum detection efficiency of 10.9+/-0.1% was measured for (228)Ac by Cherenkov counting with a very low Cherenkov photon background of 0.317+/-0.013cpm. The addition of sodium salicylate into the sample counting vial at a concentration of 0.1g/mL yielded a more than 3-fold increase in the Cherenkov detection efficiency of (228)Ac to 38%. Tests of the Cherenkov counting technique were conducted with several water standards of known activity and the results obtained compared closely with a conventional liquid scintillation counting technique. The advantages and disadvantages of Cherenkov counting compared to liquid scintillation counting methods are discussed. Advantages include much lower Cherenkov background count rates and consequently lower minimal detectable activities for (228)Ra and no need for expensive environmentally unfriendly liquid scintillation cocktails. The disadvantages of the Cherenkov counting method include the need to measure (228)Ac Cherenkov photon detection efficiency and optimum Cherenkov counting volume, which are not at all required when liquid scintillation analysis is used.