Minimal hepatic encephalopathy runs a fluctuating course: results from a three-year prospective cohort follow-up study.

INTRODUCTION: Minimal hepatic encephalopathy (mHE) has been reported in up to 84 percent of cirrhotics. The natural history of mHE has not been well-described. We designed a three-year prospective cohort study to determine the prevalence and natural history of mHE among cirrhotic patients. METHODS: The patient cohort comprising 62 consecutive outpatients with cirrhosis were assessed at baseline and followed-up with a repeat assessment three years later. The assessments include: (1) Neuropsychometric analysis (digit-symbol substitution test, block-design test, number-connection test A); (2) Clinical, biochemical assessment; and (3) Quality of life (QOL) assessment (abbreviated sickness impact profile). RESULTS: Baseline characteristics were: age 52.9 +/- 11.0 years; Child's A:B:C was 46:14:2. mHE was detected in 33.9 percent of the cohort. Older age, a higher Child-Pugh score and female gender were independently associated with mHE. mHE was associated with a poorer QOL. Follow-up assessment three years later showed that seven patients had died, while six were lost to follow-up; these patients had significantly higher baseline Child's scores. Of the remaining patients, 36/49 (73 percent) agreed to a repeat evaluation. In this group, none had mHE. QOL remained impaired despite the resolution of mHE. CONCLUSION: It has been shown for the first time that mHE can revert to a normal state in a significant proportion of patients with well-compensated cirrhosis.

Functional dissociation of DeltaPsim and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis.

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

Perforin-mediated cytotoxicity is critical for surveillance of spontaneous lymphoma.

Immune surveillance by cytotoxic lymphocytes against cancer has been postulated for decades, but direct evidence for the role of cytotoxic lymphocytes in protecting against spontaneous malignancy has been lacking. As the rejection of many experimental cancers by cytotoxic T lymphocytes and natural killer cells is dependent on the pore-forming protein perforin (pfp), we examined pfp-deficient mice for increased cancer susceptibility. Here we show that pfp-deficient mice have a high incidence of malignancy in distinct lymphoid cell lineages (T, B, NKT), indicating a specific requirement for pfp in protection against lymphomagenesis. The susceptibility to lymphoma was accentuated by simultaneous lack of expression of the p53 gene, mutations in which also commonly predispose to human malignancies, including lymphoma. In contrast, the incidence and age of onset of sarcoma was unaffected in p53-deficient mice. Pfp-deficient mice were at least 1,000-fold more susceptible to these lymphomas when transplanted, compared with immunocompetent mice in which tumor rejection was controlled by CD8(+) T lymphocytes. This study is the first that implicates direct cytotoxicity by lymphocytes in regulating lymphomagenesis.

Differential tumor surveillance by natural killer (NK) and NKT cells.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset-depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1(+) T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3(-) NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I-deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jalpha281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or alpha-galactosylceramide.

Perforin is a major contributor to NK cell control of tumor metastasis.

We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in perforin-deficient mice, suggesting that perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.

Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1.

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.

Expression of recombinant human Met-ase-1: a NK cell-specific granzyme.

Human Met-ase-1 is a member of a family of cytotoxic lymphocyte serine proteases (granzymes), but is expressed specifically in CD3- large granular lymphocytes with natural killer cell activity. We have devised a polymerase chain reaction strategy to delete the predicted hexapropeptide of human Met-ase-1 (Ser-6 to Gln-1), to enable its expression and activation in mammalian COS cells. In addition, using peptide immunization we have derived a unique and specific monoclonal antibody detecting human Met-ase-1. Western blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the human Met-ase-1 gene encodes a serine proteinase that specifically hydrolyzes substrates containing a methionine (Met-) side chain at P1. The expression of active human Met-ase-1 and the generation of a specific anti-human Met-ase-1 monoclonal antibody will now enable a detailed structure/function analysis of key amino acids that confer this unusual serine protease specificity.

Expression of recombinant human granzyme B. A processing and activation role for dipeptidyl peptidase I.

Human granzyme B (hGrzB) is the key member of a family of granule serine proteases (granzymes) that participate in target cell death inflicted by cytotoxic lymphocytes. The proenzyme activation peptide predicted from the cDNA encoding hGrzB is composed of two residues. We have devised a PCR strategy to delete this activation dipeptide within hGrzB and express active recombinant hGrzB in mammalian COS cells. Lysates of COS cells transfected with modified hGrzB cDNA were able to hydrolyze tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (Boc-Ala-Ala-Asp-SBzl), whereas lysates transfected with unmodified hGrzB cDNA were inactive. Accordingly, active recombinant hGrzB displayed no significant activities toward substrates containing Met-, Lys-, or Phe- at P1. It has been suggested that the mechanism by which the amino-terminal dipeptide is normally cleaved to generate active GrzB involves dipeptidyl peptidase I (DPPI). Our studies demonstrated that lysates of COS cells cotransfected with unmodified hGrzB cDNA (inactive) and rat DPPI cDNA were able to hydrolyze Boc-Ala-Ala-Asp-SBzl. Similarly, lysates of COS cells transfected with unmodified hGrzB cDNA, and devoid of Asp-ase activity, were also activated upon the addition of bovine spleen DPPI in a pH and time-dependent fashion. These results suggest that the activation dipeptide, and more particularly DPPI, may play and important role in the normal post-translational processing and activation of hGrzB in cytotoxic lymphocytes.

Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5'-flanking sequences.

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30,000 M(r) serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5'-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5'-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5'-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5'-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines. The transcriptional activity of the RNK-Met-1 5'-flanking region was strong, restricted to the RNK-16 LGL leukemia and controlled by several positive cis-acting regions spread over at least 3.3 kb. The longest and most active 5'-flanking region (-3341 to -33) was also used to drive specific expression of beta-galactosidase in RNK-16. These data are consistent with the NK cell-specific expression of RNK-Met-1 and suggest the potential utility of this gene promoter in the development of transgene models of NK cell biology in vivo.

Hypothesis: cytotoxic lymphocyte granule serine proteases activate target cell endonucleases to trigger apoptosis.

Upon interaction with target cells, cytotoxic T lymphocytes and natural killer cells vectorially secrete highly specialized cytoplasmic granules containing perforin and a family of serine proteases (granzymes). This granule exocytosis mechanism of cytolysis is of patho-physiological importance, and usually results in target cell DNA fragmentation. Neither perforin nor granzymes possess inherent nuclease activity, but in combination they can induce target cell apoptosis. Perforin forms transmembrane pores in the target cell, thereby enabling granzymes to access target cell substrates. The target cell substrates of granzymes are unknown, but granzyme A binding and cleavage of the nuclear shuttle protein nucleolin in target cells demonstrates that granzymes may act on nuclear substrates. Furthermore, the presence of granzyme B and other granzyme activities in the nucleus of cytotoxic lymphocytes indicates that granzymes can be transported from the cytoplasm to the nucleus. It is hypothesized that perforin enables effector granzymes to enter the target cell cytoplasm and following their transport into the nucleus, granzymes cleave specific target cell nuclear proteins to activate autolytic endonucleases that fragment DNA. In cytotoxic effectors, these nuclear substrates are normally protected from granzymes by endogenous inhibitors.

Expression of human perforin in a mouse cytotoxic T lymphocyte cell line: evidence for perturbation of granule-mediated cytotoxicity.

Expression of the pore-forming protein perforin is normally restricted to the cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Perforin, which causes cell death by osmotic lysis, has the ability to form transmembrane channels in target cell membranes. This function makes perforin crucial in the granule-exocytosis model of T cell-mediated cytotoxicity. In the present study, variants of the mouse cytotoxic T lymphocyte cell line CTLL-R8 have been produced which express human perforin. A full-length cDNA clone (HP-10) encoding human perforin was inserted in the sense orientation into the expression plasmid pCMV5neo. The resultant construct, designated pCMV5neoHP-10, was used to transfect CTLL-R8 cells. Of eight G418-resistant clones studied, four clones expressed human perforin mRNA by Northern analysis and three of these clones also expressed human perforin protein by Western blotting. The expression of human perforin protein was associated with a pronounced (55-74%) and consistent reduction in the killing of three target cell lines, P815, YAC-1, and EL4, compared with parental CTLL-R8 cells. The reduction in target cell lysis could not be attributed to nonspecific effects of the transfection, as clones transfected with neo alone showed no reduction in killing in comparison with parental CTLL-R8 cells. Clones expressing human perforin showed very similar growth characteristics, surface phenotype, and N-alpha-benzyloxycarbonyl-l-thiobenzyl-esterase release compared with untransfected CTLL-R8 cells. The mechanism of reduction of cytolysis is unclear but may involve competition by human perforin in the handling or packaging of endogenous granule constituents (including mouse perforin) or assembly of human perforin into mouse polyperforin channels in target cell membranes. The expression of human perforin in mouse cytotoxic T cells provides a potential model for studying how cytotoxic T cells process, package, utilize, and protect themselves against the perforin molecules they produce.