Strategies for clinical implementation of TNM-Immunoscore in resected nonsmall-cell lung cancer.

Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates and appears highly promising as a supplement to the tumor-node-metastasis (TNM) classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor-infiltrating lymphocytes (TILs) have been proposed by an international working group. Regardless of promising results, there are potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In nonsmall-cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC.

Low Protein A20 in Minor Salivary Glands is Associated with Lymphoma in Primary Sjögren's Syndrome.

Patients with primary Sjögren's syndrome (pSS) have an increased risk of developing lymphomas, particularly the subtype mucosa-associated lymphoid tissue (MALT) lymphoma. Chronic antigen stimulation and increased activation of nuclear factor-κB (NF-κB) are important factors for the pathogenesis of MALT lymphomas. Protein A20 is an inhibitor of NF-κB. A recent study of pSS-associated MALT lymphomas identified potential functional abnormalities in the TNFAIP3 gene, which encodes protein A20. The present study aimed to assess protein A20 by immunohistochemistry (IHC) in minor salivary glands (MSGs) and lymphoma tissue sections of patients with pSS and investigate a potential association with lymphoma development. Protein A20 staining in lymphocytes was scored in four categories (0 = negative, 1 = weak, 2 = moderate and 3 = strong). For statistical purposes, these scores were simplified into negative (scores 0-1) and positive (scores 2-3). We investigated associations between protein A20-staining, focus scores, germinal centre (GC)-like structures and monoclonal B-cell infiltration in MSGs. MSG protein A20 staining was weaker in pSS patients with lymphomas than in those without lymphomas (P = 0.01). Weak protein A20 staining was also highly associated with a lack of GC formation (P < 0.01). Finally, weaker A20 staining was observed in the majority of pSS-associated MALT lymphoma tissues. In conclusion, we found absent or weak protein A20 immunoreactivity in MSGs of patients with pSS with lymphomas. This finding indicates that protein A20 downregulation in lymphocytes might be a mechanism underlying lymphoma genesis in patients with pSS.

On the presence of a heat-stable, macromolecular inhibitor of the thrombin-fibrinogen reaction in rat liver microsomes and its separation from prothrombin.

By concentrating sonicates from rat liver microsomes containing prothrombin, the activity as measured by the one-stage prothrombin assay (Hjort, P., Rapaport, S. J. and Owren, P. A.(1965), J. Lab. Clin. Med. 46, 89-97) gradually decreased. Nearly a complete loss of prothrombin activity was found in sonicates being concentrated 10-fold. By adsorption of prothrombin on barium citrate and dissolving the precipitate in a solution of sodium citrate. NaCl and EDTA followed by gel filtration on a Sephadex G-50 column, the inhibitory effect on the bioassay of prothrombin disappeared. An inhibitor of the thrombin-fibrinogen reaction could be isolated from the supernatant after adsorption of prothrombin on barium citrate. The inhibitor was excluded from a Sephadex G-50 column equilibrated with 4 M NaCl in veronal buffer (pH 7.4) and was heat stable (70 degrees C for 10 min). No proteolytic or antitrypsin activity could be detected in the inhibitor preparation. The importance of removing the inhibitor of the microsomal sonicates prior to any bioassay of coagulation factors based on the thrombin-fibrinogen reaction is emphasized.

A study of intracellular transport of secretory glycoproteins in rat liver.

To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.

Vitamin K epoxidase activity of rough and smooth microsomes from rat liver.

The distribution of vitamin K epoxidase activity in rough and smooth microsomes has been studied and compared to the prothrombin precursor and vitamin K-dependent carboxylase activity. All three activities were high in rough microsomes as compared to the low levels found in smooth microsomes. The results are in agreement with the suggestion that there might be a linkage between the vitamin K-dependent carboxylation and epoxidation reaction in vivo.

Biological and immunological activity of prothrombin in rough and smooth microsomes isolated from rat liver.

1. The presence of biological and immunological activity of prothrombin in sonicates from rough and smooth microsomes has been investigated. 2. Biological activity of prothrombin was detected in both the rough and smooth microsomal fraction. The specific and the total activity of prothrombin in the sonicates from smooth microsomes were 3-4-fold higher than in the corresponding fraction from rough microsomes. 3. Prothrombin could be identified in both microsomal fractions by double immunodiffusion. 4. The presence of a macromolecular inhibitor of blood coagulation in the microsomes is reported.

Biosynthesis and clearance of prothrombin in warfarin-treated rats.

The steady-state concentration of abnormal plasma prothrombin in warfarin-treated rats (10 mg/kg) was found to be approx. 6% of the plasma prothrombin level in normal rats. The clearance of abnormal plasma prothrombin in warfarin-treated rats was studied using either cycloheximide, to inhibit the synthesis, or vitamin K, to block the appearance of abnormal prothrombin in plasma. The clearance of abnormal plasma prothrombin corresponded to a half-life of approx. 6 h, which is similar to the half-life of normal plasma prothrombin. The de novo synthesis of prothrombin in warfarin-treated and normal rats was compared by measuring the incorporation of [3H]leucine into plasma prothrombin 90 min after an intravenous injection of the isotope. In warfarin-treated rats, accumulated prothrombin precursor was carboxylated and transported into circulation by injecting vitamin K 30 min after isotope administration. On comparing the incorporation of [3H]leucine into plasma prothrombin in warfarin-treated and normal rats, no significant difference in the de novo synthesis was detected. Our results suggest that the secretion of prothrombin in warfarin-treated rats is decreased to 6% of the normal rate. As the de novo synthesis is not affected by warfarin treatment, more than 90% of the newly synthesized prothrombin appears to be degraded intracellularly.

A study on the intracellular transport of prothrombin, albumin and transferrin in rat.

The intracellular transport of prothrombin in rat has been studied and compared with the transport of albumin and transferrin. The proteins were immunoisolated from plasma samples after pulse labelling with [3H]leucine and the secretion kinetics were determined. The half-times for secretion (t1/2) were approx. 30, 53 and 75 min for albumin, prothrombin and transferrin, respectively, whereas the minimal transit time for prothrombin was approx. 30 min, and those for albumin and transferrin 15-20 min. After injection of vitamin K-1 into warfarin-treated rats, the accumulated prothrombin precursor was gamma-carboxylated and secreted with a t1/2 of 37 min. This indicates that the gamma-carboxylation of prothrombin in rough endoplasmic reticulum cannot account for the delay in the transport of prothrombin as compared to albumin. Comparison of the incorporation of [3H]leucine and [3H]glucosamine into plasma prothrombin and transferrin suggested that transferrin is secreted randomly from an intracellular pool, whereas prothrombin is transported in a more orderly sequence. Moreover, treatment of rough microsomes with 0.05% sodium deoxycholate indicated that prothrombin is more tightly associated with the membranes of rough endoplasmic reticulum than albumin and transferrin.

On the significance of the carbohydrate moieties of bovine prothrombin for clotting activity.

Purified bovine prothrombin has been treated with different mixtures of glycosidases. Upon incubation of the prothrombin for 30 h with a combination of neuraminidase, alpha- and beta-galactosidase and beta-N-acetylglucosaminidase in 4 mM diisopropylfluorophosphate at pH 5.3 and 30 degrees C, about 70% of the carbohydrates were removed without affecting the coagulation activity. All the sialic acid and about half of the mannose, galactose and glucosamine residues were removed by this treatment.

Saponin permeabilization of rough microsomes from rat liver reveals a novel prothrombin pool.

Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following (14)C-gamma-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.

Investigation of a possible correlation between rates of secretion and microsomal membrane association of plasma proteins synthesized by rat liver.

The rates of secretion of complement C3, haptoglobin and plasminogen have been determined after pulse labelling with [3H]leucine, and compared to the secretion of prothrombin, albumin and transferrin investigated previously (Kvalvaag, A.H., Tollersrud, O.K. and Helgeland, L. (1988) Biochim. Biophys. Acta 937, 319-327). To study membrane association, rough microsomes were treated with increasing concentrations of saponin, sodium deoxycholate or Triton X-100. All six proteins were quantitated in the soluble and membrane fraction by enzyme immunoassays. At concentrations of saponin from 0.08% to 0.32%, each secretory protein showed a characteristic distribution, almost identical to that obtained with 0.05% sodium deoxycholate or 0.08% Triton X-100. Albumin and transferrin with half-times for secretion (t1/2) 30 and 75 min, respectively, are both almost exclusively found in the luminal fraction (greater than 95%). Prothrombin and plasminogen, which both show an intermediate t1/2 (approx. 55 min), are partially associated with the membranes, as only about 60% was released. Haptoglobin and complement C3 also show some association with the membranes (80-85% released). C3 is secreted at the same rate as prothrombin and plasminogen (t1/2 = 55 min), whereas haptoglobin is secreted more rapidly (t1/2 = 40 min). Accordingly, no correlation between kinetics of secretion and membrane association was demonstrated.

The time courses of intracellular transport of some secretory proteins of rat liver are not affected by an induced acute phase response.

To study the effects of changed synthesis on intracellular transport of secretory proteins, an acute phase response was induced in rats. The synthesis and secretion of haptoglobin, complement C3, transferrin and albumin were then investigated by pulse labeling with [3H]leucine. Maximal increase in the syntheses of the positive acute phase proteins was observed after 24 h, amounting to an increase of nine, three and twofold for haptoglobin, C3 and transferrin, respectively. The synthesis of albumin decreased to a minimum after 48 h, reaching approximately one fourth of normal synthesis. The time courses for transit through rough endoplasmic reticulum and for secretion were determined after 36 h, and were found to be roughly unchanged for all four proteins despite the different changes in synthesis. The fraction of haptoglobin associated with the microsomal membrane was reduced during the acute phase response, but there was no significant change in membrane association as a function of time after labeling with [3H]leucine. It is concluded that the altered protein synthesis during an acute phase response in vivo has little effect on the time course of secretion of the proteins studied. Furthermore, the basal mechanisms for intracellular transport appear relatively unchanged during this condition.

Studies of an inhibitor of the thrombin-fibrinogen reaction localized in rat liver microsomes. Interference with the polymerization step.

A heat-stable, macromolecular inhibitor of the thrombin-fibrinogen reaction localized in rat liver microsomes has been shown to interfere with the polymerization step in the fibrinogen-fibrin conversion. The inhibitor had no effect on thrombin activity as measured with the synthetic, chromogenic substrate BZ-Phe-Val-Arg-pNA. The amount of fibrin formed and the release of fibrinopeptide A were not affected by the inhibitor. Recording of turbidity at 350 nm and 600 nm indicated an inhibition of the lateral aggregation of the end-to-end fibrin polymers. The inhibitor was localized in both the luminal and membrane fractions of the microsomes. The inhibitor activity was not affected by warfarin treatment of the rats.

On the quantification of prothrombin from different species using Echis carinatus as activator.

The generation of thrombin-like activity from rat, human, bovine and mouse prothrombin by Echis carinatus venom (ECV) treatment was compared using a partially purified system (i.e. whole ECV and isolated prothrombin). A rapid increase in coagulant activity was obtained within 0.5 to 2 min., being constant upon further incubation for 60 min. A large variation in coagulant activity of the ECV generated thrombin from the four species was found, whereas no differences were found for the amidolytic activities. The coagulant activities of the ECV generated thrombin was also low compared with the corresponding thrombin activities obtained by physiological activation. Coagulant activity of the ECV generated thrombin levelled off at increasing concentration of prothrombin in the sample as measured by the one-stage coagulation assay. By measuring amidolytic activity a linear relationship to the concentration of prothrombin was found, however. These findings indicate that ECV converts prothrombin from the four different species to a thrombin-like protein with properties distinct from alpha-thrombin. The lack of linearity in the ECV generated clot activity with increasing concentration of prothrombin could be explained by assuming a dimerization of the thrombin-like protein molecules making them less accessible to fibrinogen. The significance of these observations for the quantification of prothrombin from different species is discussed.

Indications of 'atopic bowel' in patients with self-reported food hypersensitivity.

BACKGROUND: An association between atopic disease and gastrointestinal complaints has been suggested. AIM: To explore the association between atopic disease, gastrointestinal symptoms, and possible gastrointestinal manifestations of atopic disease in patients with self-reported food hypersensitivity. METHODS: Symptoms, skin prick tests, serum markers of allergy and intestinal permeability were recorded in 71 adult patients. Eosinophils, tryptase- and IgE-positive cells were counted in duodenal biopsies. RESULTS: Sixty-six (93%) patients had irritable bowel syndrome (IBS) and 43 (61%) had atopic disease, predominantly rhinoconjunctivitis. All 43 were sensitized to inhalant allergens, 29 (41%) to food allergens, but food challenges were negative. Serum total IgE and duodenal IgE-positive cell counts were significantly correlated (P < 0.0001) and both were significantly higher in atopic than in non-atopic patients (P < 0.0001 and P = 0.003 respectively). IgE-positive cells appeared to be 'armed' mast cells. Intestinal permeability was significantly elevated in atopic compared with non-atopic patients (P = 0.02). Gastrointestinal symptoms and numbers of tryptase-positive mast cells and eosinophils did not differ between groups. CONCLUSIONS: Patients with self-reported food hypersensitivity had a high prevalence of IBS and atopic disease. Atopic patients had increased intestinal permeability and density of IgE-bearing cells compared with non-atopic patients, but gastrointestinal symptoms did not differ between groups.

Effect of warfarin on ATP content, viability, glycosylation and protein synthesis in isolated rat hepatocytes.

The effect of warfarin on ATP content, viability and incorporation of labelled glucosamine and leucine into protein was studied in suspensions of rat hepatocytes. When incubated with warfarin the synthesis of total protein and glycoprotein was equally inhibited, and the viability and ATP content of the cells were also lowered. These observations suggest that warfarin is not an inhibitor of glycosylation of proteins in isolated hepatocytes, however, the presence of warfarin leads to reduced ATP content of the cells which causes a decrease in protein synthesis and viability.