Transcriptomic analysis of hepatocellular carcinoma reveals molecular features of disease progression and tumor immune biology.

Hepatocellular carcinoma (HCC) develops in the context of chronic inflammatory liver disease and has an extremely poor prognosis. An immunosuppressive tumor microenvironment may contribute to therapeutic failure in metastatic HCC. Here, we identified unique molecular signatures pertaining to HCC disease progression and tumor immunity by analyzing genome-wide RNA-Seq data derived from HCC patient tumors and non-tumor cirrhotic tissues. Unsupervised clustering of gene expression data revealed a gradual suppression of local tumor immunity that coincided with disease progression, indicating an increasingly immunosuppressive tumor environment during HCC disease advancement. IHC examination of the spatial distribution of CD8+ T cells in tumors revealed distinct intra- and peri-tumoral subsets. Differential gene expression analysis revealed an 85-gene signature that was significantly upregulated in the peri-tumoral CD8+ T cell-excluded tumors. Notably, this signature was highly enriched with components of underlying extracellular matrix, fibrosis, and epithelial-mesenchymal transition (EMT). Further analysis condensed this signature to a core set of 23 genes that are associated with CD8+ T cell localization, and were prospectively validated in an independent cohort of HCC specimens. These findings suggest a potential association between elevated fibrosis, possibly modulated by TGF-ß, PDGFR, SHH or Notch pathway, and the T cell-excluded immune phenotype. Indeed, targeting fibrosis using a TGF-ß neutralizing antibody in the STAM™ model of murine HCC, we found that ameliorating the fibrotic environment could facilitate redistribution of CD8+ lymphocytes into tumors. Our results provide a strong rationale for utilizing immunotherapies in HCC earlier during treatment, potentially in combination with anti-fibrotic therapies.

Up-regulation of LFA-1 allows liver-resident memory T cells to patrol and remain in the hepatic sinusoids.

Liver-resident CD8+ T cells are highly motile cells that patrol the vasculature and provide protection against liver pathogens. A key question is: how can these liver CD8+ T cells be simultaneously present in the circulation and tissue-resident? Because liver-resident T cells do not express CD103 - a key integrin for T cell residence in epithelial tissues - we investigated other candidate adhesion molecules. Using intra-vital imaging we found that CD8+ T cell patrolling in the hepatic sinusoids is dependent upon LFA-1-ICAM-1 interactions. Interestingly, liver-resident CD8+ T cells up-regulate LFA-1 compared to effector-memory cells, presumably to facilitate this behavior. Finally, we found that LFA-1 deficient CD8+ T cells failed to form substantial liver-resident memory populations following Plasmodium or LCMV immunization. Collectively, our results demonstrate that it is adhesion through LFA-1 that allows liver-resident memory CD8+ T cells to patrol and remain in the hepatic sinusoids.

A role for FOXO1 in BCR-ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired ABL1 kinase domain mutations are successfully treated with second-generation ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or ponatinib. However, ~40% of relapsed patients have uncharacterized BCR-ABL1 kinase-independent mechanisms of resistance. To identify these mechanisms of resistance and potential treatment options, we generated ABL-TKI-resistant K562 cells through prolonged sequential exposure to imatinib and dasatinib. Dual-resistant K562 cells lacked BCR-ABL1 kinase domain mutations, but acquired other genomic aberrations that were characterized by next-generation sequencing and copy number analyses. Proteomics showed that dual-resistant cells had elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells, resistant cells were sensitive to growth inhibition and apoptosis induced by the class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear translocation. FOXO1 was elevated in a subset of primary specimens from relapsed CML patients lacking BCR-ABL1 kinase domain mutations, and these samples were responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1 contributes to BCR-ABL1 kinase-independent resistance experienced by these CML patients and that PI3K inhibition coupled with BCR-ABL1 inhibition may represent a novel therapeutic approach.

Local repeat sequence organization of an intergenic spacer in the chloroplast genome of Chlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: a complex mode of "copy-choice replication"?

Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA of Chlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt+) in a cross between a pair of polymorphic interfertile strains of Chlamydomonas (C. reinhardtii and C. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, "P2" (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3' end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, "P2" seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely "sequence-scrambled" product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a "unique" new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a "complex path" of copy-choice replication.

A self-consistent cell flux expression for simultaneous chemotaxis and contact guidance in tissues.

During wound healing, both chemotaxis and contact guidance can contribute to the migration of blood and tissue cells to the wound. In order to understand the wound healing process, we must thus understand how cells respond to both these simultaneous directional cues, which are not necessarily coaligned. Although chemotaxis and contact guidance have been studied individually, the interaction between them has not been addressed. We extend a stochastic cell movement model, developed by Dickinson and Tranquillo (1995) [6] for individual cues, for simultaneous chemotaxis and contact guidance by a two-parameter perturbation analysis in terms of the two associated cues, a chemotactic factor gradient and aligned tissue fibers. We present results from analysis of the first-order perturbation, which includes the cell flux expression heuristically proposed by others, but reveals paradoxical results for other indices of cell movement, such as the mean-squared displacement. We then present second-order perturbation results that resolve these paradoxical results. Finally, we relate these results to a continuum mechanical model developed by Barocas and Tranquillo (1997) [3] that predicts fiber alignment due to cell traction induced tissue contraction.

RecA interacts with Klenow and enhances fidelity of DNA synthesis in vitro.

To understand the molecular basis of RecA-mediated DNA-repair, we tested the replicative fidelity of the large fragment of Pol I (Klenow) in RecA-DNA complexes in vitro. Klenow synthesis was error-prone in naked DNA substrates but essentially error-free in RecA coated complexes. Escherichia coli SSB, causes no such improvement in Klenow fidelity. RecA filaments promote better exonucleolytic proofreading by Klenow than on naked DNA substrates at select sites when replication is "stalled" due to a missing dNTP. Addition of RecA to pyrene sulfonylchloride-labeled Klenow resulted in a specific increase in steady-state fluorescence anisotropy and a concomitant decrease in fluorescence lifetime. These observations suggest the possibility of a direct interaction between RecA and Klenow even in the absence of DNA which may mediate the observed improvement in Klenow fidelity.

Non-Watson-Crick base pairs modulate homologous alignments in RecA pairing reactions.

Complementary pairing by RecA was examined in vitro to investigate how homology is deciphered from non-homology. Somewhere in a window of 40-50% sequence complementarity, RecA pairing begins to manifest the specificity of homology. Quantitation reveals a hierarchy among non-Watson-Crick mispairs: RecA reaction treats six out of 12 possible mispairs as good ones and three each of the remaining ones as moderate and bad pairs. The mispairs seem to function as independent pairing units free of sequence context effects. The overall strength of pairing is simply the sum of the constituent units. RecA mediated gradation of mispairs, free of sequence context effects, might offer a general thumb-rule for predicting the pairing strength of any alignment that carries multiple mispairs.

Influence of triple-lumen central venous catheters coated with chlorhexidine and silver sulfadiazine on the incidence of catheter-related bacteremia.

OBJECTIVE: To evaluate the efficacy of triple-lumen central venous catheters coated with a combination product of chlorhexidine and silver sulfadiazine (CSS) in reducing the incidence of local catheter infection and catheter-related bacteremia. DESIGN: Randomized, controlled trial. SETTING: The surgical intensive care units in a university hospital. PATIENTS: All patients who needed central venous catheterization were randomized to receive either an uncoated triple-lumen catheter (n = 157) or a catheter coated with CSS (n = 151). MAIN OUTCOME MEASURE: Catheters were removed when no longer needed or suspected as a cause of infection. The tip and a 5-cm segment of the intradermal portion of the catheter were cultured semiquantitatively. Blood cultures were obtained when clinically indicated. The remaining segment of catheters coated with CSS were cut and incubated on an agar plate with strains of Staphylococcus aureus and Enterococcus. Zone of inhibition was determined 24 hours later. Data were analyzed by survival and logistic multivariate regression methods. RESULTS: Catheters coated with CSS were effective in reducing the rate of significant bacterial growth on either the tip or intradermal segment (40%) compared with control catheters (52%; P = .04). However, there was no difference in the incidence of catheter-related bacteremia (3.8% [uncoated] vs 3.3% [coated]; P = .81). In vitro activity of catheters with CSS against S aureus was evident up to 25 days but activity against Enterococcus dissipated more quickly over time and was absent by day 4. The most common colonizing organisms were coagulase-negative staphylococcus and enterococcus. Variables that were associated with a significant amount of growth on the tip or intradermal segment were a duration of catheterization of longer than 7 days, jugular insertion site, and the absence of a CSS coating. The use of a guidewire when the catheter was removed was associated with a lower risk of significant bacterial growth. CONCLUSIONS: The use of CSS reduces the incidence of significant bacterial growth on either the tip or intradermal segments of coated triple-lumen catheters but has no effect on the incidence of catheter-related bacteremia. In this patient population, catheters coated with CSS provide no additional benefit over uncoated catheters.

Endoscopic placement of nasoenteric feeding tubes in critically ill patients: a reliable alternative.

Spontaneous transpyloric passage of nasoenteric feeding tubes is often unsuccessful in critically ill patients due to gastric ileus. The purpose of this study was to evaluate the success rate of endoscopic placement of postpyloric nasoenteric tubes and the time required to achieve goal enteral nutritional support in critically ill patients with gastric ileus. Surgical ICU patients with gastric ileus, documented by recorded high gastric residual volumes via a nasogastric tube, were identified for placement of a nasoenteric postpyloric tube via esophagogastroduodenoscopy (EGD). EGD was performed bedside in the ICU, using intravenous sedation, for placement of a 43-inch (109 cm) 8 French tungsten-weighted nasoenteric tube with an inner stylet into the duodenum, and the tube was advanced as far distally as possible. Abdominal radiograph was obtained to confirm final tube position postprocedure. Enteral feedings were initiated immediately with a full-strength formula and increased to the goal enteral feeding rate as tolerated. Thirty-four patients underwent successful placement of postpyloric nasoenteric tubes by EGD. One procedure was aborted in a patient with adult respiratory distress syndrome (ARDS) who developed hypoxemia with gastric insufflation during the EGD. The mean time to achievement of goal enteral nutritional support was 20.8 hours. Tubes remained in place for a range of 6 to 37 days. Endoscopic placement of postpyloric enteral feeding tubes is highly successful, and allows for prompt achievement of goal enteral nutritional requirements. It has two main advantages: it eliminates the risk of patient travel to radiology for fluoroscopic placement, and allows for earlier initiation of enteral feedings because spontaneous passage of weighted nasoenteric tubes into the duodenum in critically ill patients is often unsuccessful.

Spontaneous splenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): occurrence in an allogeneic donor of peripheral blood stem cells.

Granulocyte colony-stimulating factor (G-CSF) has been used to improve granulocyte count in chronic neutropenia and myelodysplasia, to minimize the incidence and duration of neutropenia during conventional chemotherapy, and to mobilize peripheral blood stem cells prior to leukapheresis for use in autologous and allogeneic marrow transplantation. The most common toxicity is bone pain, and other reactions such as inflammation at the site of injection have also occurred. In patients with chronic neutropenia, splenomegaly has been described with long-term use, and extramedullary hematopoiesis has also been reported. However, thus far, no life-threatening sequelae of these effects are found in the literature. We now describe a case of spontaneous splenic rupture four days following a six-day course of G-CSF therapy in an allogeneic donor of peripheral blood stem cells.

A comparison of 10% pentastarch and 5% albumin in patients undergoing open-heart surgery.

Colloids are useful in cardiac surgery to increase preload and improve cardiac output without the risks associated with blood transfusions. Pentastarch is a new low-molecular weight hydroxyethyl starch compound under investigation for this purpose. The authors compared, in a randomized fashion, 12 patients who received pentastarch and 17 patients who received albumin for volume expansion after open-heart surgery. During the 24-hour study period there was no significant difference between the two groups with respect to systemic blood pressure, mean arterial pressure, cardiac index, right atrial pressure, and pulmonary capillary wedge pressure, with the exception of a higher mean arterial pressure and systolic blood pressure at 4 hours in the albumin group and higher heart rate at 12 hours in the pentastarch group. In addition, postoperative prothrombin time, partial thromboplastin time, fibrinogen, platelets, and factor VIII levels were not significantly different between the two groups. There were no complications attributed to colloid administration. The hemodynamic parameters were further evaluated in a subset of 6 pentastarch and 9 albumin patients who received the first 500 mL of colloid in a similar time frame and under similar clinical conditions. The patients who received pentastarch showed a significantly greater increase in cardiac index than did the patients who received albumin. No significant change in other parameters were noted between the two groups. The authors conclude that pentastarch is as safe as albumin and may be a more effective volume expander than albumin when used in open-heart surgery patients.