OBJECTIVE

Population-based studies for the prevalence of coeliac disease (CD) in west-Asian countries are scarce. We aimed to determine the prevalence of gluten-sensitive enteropathy (GSE) in the general population of northern and southern Iran, and evaluate the sensitivity and specificity of the anti-endomysial antibody (EMA) immunofluorescent test and the enzyme-linked immunosorbent assay-based test for determination of the IgA anti-tissue transglutaminase antibody (tTG-Ab) using the human recombinant transglutaminase antigen for the detection of CD in screening the asymptomatic adult population.

METHODS

Using a stratified random sampling method we enrolled a total of 2799 individuals (1438 from Sari and 1361 from Kerman). The mean age was 33.7 years (range 18-66), with 1398 men. IgA anti-tissue transglutaminase (tTG) and IgA anti-EMA were determined in the serum of all subjects. Those participants with a positive serology for any of the two tests underwent small intestinal biopsy, and were classified according to revised Marsh criteria histologically. A diagnosis of GSE was based on positive serology and a compatible histopathological finding. The maximum likelihood latent class model was used to estimate the sensitivity and specificity of the two tests.

RESULTS

Twenty-nine cases showed positive IgA tTG-Ab (15 men and 14 women, mean age 35.4 years, range 18-59), whereas only five were simultaneously positive for EMA. Except for two subjects with normal small bowel histology (Marsh 0), all other subjects were found to have biopsy findings compatible with GSE: 18 Marsh I, five Marsh II, three Marsh IIIa and one Marsh IIIc lesions. he prevalence of GSE was 0.96% or 1:104. The sensitivity and specificity of the human-recombinant IgA tTG-Ab assay were 100 and 99%, respectively, whereas the results for IgA EMA were 19 and 100%, respectively. The IgA EMA was positive in cases with advanced mucosal lesions of the small bowel. The mean serum value of IgA tTG-Ab was higher in patients with severe enteropathy compared with those showing slight mucosal changes (P<0.05).

CONCLUSIONS

The minimum prevalence of gluten sensitivity among the general population of northern and southern Iran is 1:104. The best screening test for the detection of GSE in the general population is IgA tTG-Ab.

The simian virus 40 agnoprotein is a 61-amino-acid, highly basic polypeptide that is coded within the 5' leader of late 16S mRNAs. To better understand agnoprotein function and to more effectively differentiate cis-from trans-acting effects of an agnogene mutation, we constructed a mutant virus that carries a single-base-pair substitution and fails to produce agnoprotein. pm 1493 contains a T/A to A/T transversion at sequence position 335. This mutation converts the agnoprotein initiation codon from ATG to TTG, preventing synthesis of the protein. The mutant displays only a modest growth defect in CV-1P and AGMK cells and no defect in BSC-1 cells. Early-gene expression, DNA replication, synthesis of late viral products, and the kinetics of virion assembly all appear normal in pm 1493-infected CV-1P cells. Immunofluorescent studies, however, indicate that localization of the major capsid polypeptide VP1 is different in mutant- than wild-type virus-infected cells. Furthermore, the lack of agnoprotein led to inefficient release of mature virus from the infected cell. Agnogene mutants could be severely compromised in their ability to propagate in monkeys given their reduced capacity for cell-to-cell spread.

OBJECTIVE

To assess new metrics of tumor-size response to predict overall survival (OS) in colorectal cancer (CRC) in Western and Chinese patients.

METHODS

Various metrics of tumor-size response were estimated using longitudinal tumor size models and data from two phase III studies that compared bevacizumab plus chemotherapy versus chemotherapy as first-line therapy in Western (n = 923) and Chinese (n = 203) patients with CRC. Baseline prognostic factors and tumor-size metrics estimates were assessed in multivariate models to predict OS. Predictive performances of the models were assessed by simulating multiple replicas of the phase III studies.

RESULTS

Time to tumor growth (TTG) was the best metric to predict OS. TTG fully captured bevacizumab effect. Chinese ethnicity had no impact on OS or on the TTG-OS relationships. The model correctly predicted OS distributions in each arm as well as bevacizumab hazard ratio (model prediction, 0.75 v 0.68 observed in Western patients; 95% prediction interval, 0.62 to 0.91).

CONCLUSIONS

TTG captured therapeutic benefit with bevacizumab in first-line CRC patients. Chinese ethnicity had no impact. Longitudinal tumor size data coupled with model-based approaches may offer a powerful alternative in the design and analysis of early clinical studies.

Tissue transglutaminase (tTG) is a unique member of the transglutaminase family as it is both a transamidating enzyme and a GTPase. In the cell tTG is mostly cytosolic, however it is also found in the nucleus and associated with the plasma membrane. tTG can be proapoptotic, however anti-apoptotic activities of the enzyme have also been reported. To determine how the intracellular localization and transamidating activity of tTG modulates its effects on apoptosis, HEK293 cells were transiently transfected with tTG or [C277S]tTG (which lacks transamidating activity) constructs that were targeted to different intracellular compartments. Apoptosis was induced by thapsigargin treatment, which results in increased intracellular calcium concentrations. Cytosolic tTG was pro-apoptotic, while nuclear localization of [C277S]tTG attenuated apoptosis. Membrane-targeted tTG had neither pro- nor anti-apoptotic functions. This finding indicates for the first time that intracellular localization is an important determinant of the effect of tTG on apoptosis. Previous studies have suggested that tTG may modulate retinoblastoma (Rb) protein, an important suppressor of apoptosis. tTG interacted with Rb and after induction of apoptosis, the interaction of nuclear-targeted [C277S]tTG with Rb was increased significantly concomitant with an attenuation of apoptosis. In contrast, the interaction of nuclear-targeted tTG with Rb was significantly decreased and apoptosis was not attenuated. These data suggest that tTG protects cells against apoptosis in response to stimuli that do not result in increased transamidating activity by translocating to the nucleus, and that complexing with Rb may be an important aspect of the protective effects of tTG.

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage phiC31. Host strains expressing the phiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.

OBJECTIVE

To evaluate the predictive value of tissue transglutaminase (tTG) antibodies for villous atrophy in adult and pediatric populations to determine if duodenal biopsy can be avoided.

METHODS

A total of 324 patients with celiac disease (CD; 97 children and 227 adults) were recruited prospectively at two tertiary centers. Human IgA class anti-tTG antibody measurement and upper gastrointestinal endoscopy were performed at diagnosis. A second biopsy was performed in 40 asymptomatic adults on a gluten-free diet (GFD) and with normal tTG levels.

RESULTS

Adults showed less severe histopathology (26% vs 63%, P < 0.0001) and lower tTG antibody titers than children. Levels of tTG antibody correlated with Marsh type in both populations (r = 0.661, P < 0.0001). Multiple logistic regression revealed that only tTG antibody was an independent predictor for Marsh type 3 lesions, but clinical presentation type and age were not. A cut-off point of 30 U tTG antibody yielded the highest area under the receiver operating characteristic curve (0.854). Based on the predictive value of this cut-off point, up to 95% of children and 53% of adults would be correctly diagnosed without biopsy. Despite GFDs and decreased tTG antibody levels, 25% of the adults did not recover from villous atrophy during the second year after diagnosis.

CONCLUSIONS

Strongly positive tTG antibody titers might be sufficient for CD diagnosis in children. However, duodenal biopsy cannot be avoided in adults because disease presentation and monitoring are different.

An in-frame deletion of 801 bp in exons 2-7 (type III mutation) of the epidermal growth factor receptor (EGFR) is detected at high incidence in primary glioblastoma tumors. A proteomic approach was used to generate differential protein expression maps of fetal human astrocytes (FHA), human glioblastoma cell lines U87MG and U87MG expressing type III EGFR deletion (U87MGdeltaEGFR) that confers high malignancy to tumor cells. Two-dimensional gel electrophoresis followed by in-gel digestion of separated spots and protein identification by LC-MS-MS and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified 23 proteins expressed at higher levels or exclusively in FHA and 29 proteins expressed at higher levels or exclusively in U87MG cells. Three proteins, ubiquitin, cystatin B, and tissue transglutaminase (TTG), were upregulated in U87MGdeltaEGFR relative to U87MG. Four proteins highly expressed by U87MG cells, Hsp27, major vault protein, TTG, and cystatin B, were analyzed by Western blot, ELISA, or RT-PCR in cell extracts and in tissue samples of glioblastoma multiforme (GBM; grade IV), low-grade astrocytomas (grades I and II), and nonmalignant brain lesions. All four proteins were highly expressed in GBM tissues compared to nonmalignant brain. These proteins may be used as diagnostic or functional (e.g., multiple drug resistance, invasiveness) markers for glioblastoma tumors.

The objective of this study was to evaluate the possibility of linking the tracing of whole blood clotting in a thrombelastograph (TEG) hemostasis system with the generation of thrombin assessed by thrombin/antithrombin complex (TAT). Citrated whole blood containing corn trypsin inhibitor from volunteers was clotted in the presence of CaCl2 and tissue factor. Clotting was monitored with the eight channels of a TEG system. At different time points, the whole blood TEG reaction cups were kept in a cold quenching solution, centrifuged, and the supernatants were kept at -80 degrees C until assayed for TAT by ELISA. The total thrombus generation (TTG) was calculated from the first derivative of the TEG waveform and was compared with thrombin generation measured by TAT. The two vector values--the TAT thrombin generation data and the corresponding TEG TTG--were analyzed using Pearson correlation coefficients (r) and linear, non-linear and natural log (ln) transformation of TAT values for least-squares goodness-of-fit curves. The best least-squares fit is an exponential curve. Linearizing using the ln of the TAT thrombin generation variable produces the same r (0.94) as of the exponential curve. The prediction equation is y = 8.0465 + 0.0005x (P < or = 0.0001), where y is the TAT thrombin generation variable in the ln transformation and x is the TEG TTG variable. The high magnitude of r and the high significance of the prediction equation demonstrate the high efficacy of the prediction of TAT thrombin generation by the use of TEG TTG.

Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.

The complete nucleotide sequences of the mt genome from the firefly, Pyrococelia rufa (Coeleoptera: Lampyridae) was determined. The circular genome is 17,739-bp long, and contains a typical gene complement, order, and arrangement identical to Drosophila yacuba. The presence of 1,724-bp long intergenic spacer in the P. rufa mt genome is unique. The putative initiation codon for ND1 gene appears to be TTG, instead of frequently found ATN. All tRNAs showed stable canonical clover-leaf structure of other mt tRNAs, except for tRNA(Ser) (AGN), DHU arm of which could not form stable stem-loop structure. Phylogenetic analysis among insect orders confirmed a monophyletic Endopterygota, a monophyletic Mecopterida, a monophyletic Diptera, a monophyletic Lepidoptera, and a monophyletic Coleoptera, suggesting that the complete insect mt genome sequence has a resolving power in the diversification events within Endopterygota. However, internal relationships among three coleopteran species are not clear, and the inclusion of some insect orders (i.e., apterygotan T. gertschi) in the analysis provided inconsistent results compared to other molecular studies.

OBJECTIVE

To establish the diagnostic performance of several serological tests, individually and in combination, for diagnosing celiac disease (CD) in patients with different pretest probabilities, and to explore potential serological algorithms to reduce the necessity for biopsy.

METHODS

We prospectively performed duodenal biopsy and serology in 679 adults who had either high risk (n = 161) or low risk (n = 518) for CD. Blood samples were tested using six assays (enzyme-linked immunosorbent assay) that detected antibodies to tissue transglutaminase (tTG) and deamidated gliadin peptide (DGP).

RESULTS

CD prevalence was 39.1% in the high-risk population and 3.3% in the low-risk group. In high-risk patients, all individual assays had a high diagnostic efficacy [area under receiving operator characteristic curves (AU ROC): 0.968 to 0.999]. In contrast, assays had a lower diagnostic efficacy (AU ROC: 0.835 to 0.972) in the low-risk group. Using assay combinations, it would be possible to reach or rule out diagnosis of CD without biopsy in 92% of cases in both pretest populations. We observed that the new DGP/tTG Screen assay resulted in a surplus compared to more conventional assays in any clinical situation.

CONCLUSIONS

The DGP/tTG Screen assay could be considered as the best initial test for CD. Combinations of two tests, including a DGP/tTG Screen, might be able to diagnose CD accurately in different clinical scenarios making biopsy avoidable in a high proportion of subjects.

OBJECTIVE

A multicenter phase II trial of ranpirnase (Onconase; Alfacell Corp, Bloomfield, NJ) as a single agent was conducted to further assess the safety and clinical efficacy of this novel antitumor ribonuclease. Patients with unresectable and histologically confirmed malignant mesothelioma (MM) were eligible.

METHODS

One hundred five patients with Eastern Cooperative Oncology Group performance status 0 to 2 were enrolled onto the study. Thirty-seven percent of patients had not responded to prior chemotherapy. The primary end point of the study was survival. Tumor responses and time to progression were also assessed. The Cancer and Leukemia Group B (CALGB) prognostic group criteria were used to define a treatment target group (TTG). Both the intent-to-treat (ITT) and the TTG populations were analyzed for survival.

RESULTS

Median survival times of 6 months for the ITT and 8.3 months for the TTG populations were observed. The 1- and 2-year survival rates were 34.3% and 21.6% for ITT, respectively, and 42% and 26.8% for TTG, respectively. Among the 81 patients assessable for tumor response, four had partial responses, two had minor regressions, and thirty-five experienced stabilization of previously progressive disease. Patients with responses and stable disease demonstrated markedly prolonged survival. Ranpirnase was well tolerated in the majority of patients, and there were no drug-related deaths.

CONCLUSIONS

Ranpirnase demonstrated activity and a tolerable toxicity profile in patients with unresectable MM. The prognostic value of the CALGB groups was confirmed.

Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be used for the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems.

In order to understand the mechanism for insoluble neurotoxic protein polymerization in Alzheimer's disease (AD) brain neurons, we examined protein and gene expression for transglutaminase (TGase 2; tissue transglutaminase (tTG)) in hippocampus and isocortex. We found co-localization of tTG protein and activity with tau-positive neurofibrillary tangles, whereas mRNA and sequence analysis indicated an absolute increase in tTG synthesized. Although apoptosis in AD hippocampus is now an established mode of neuronal cell death, no definite underlying mechanism(s) is known. Since TGase-mediated protein aggregation is implicated in polyglutamine ((CAG)(n)/Q(n) expansion) disorder apoptosis, and expanded Q(n) repeats are excellent TGase substrates, a role for TGase in AD is possible. However, despite such suggestions almost 20 years ago, the molecular mechanism remained elusive. We now present one possible molecular mechanism for tTG-mediated, neurotoxic protein polymerization leading to neuronal apoptosis in AD that involves not its substrates (like Q(n) repeats) but rather the unique presence of alternative transcripts of tTG mRNA. In addition to a full-length (L) isoform in aged non-demented brains, we found a short isoform (S) lacking a binding domain in all AD brains. Our current results identify intron-exon "switching" between L and S isoforms, implicating G-protein-coupled signaling pathways associated with tTG that may help to determine the dual roles of this enzyme in neuronal life and death processes.

In Parkinson's disease (PD), conformational changes in the alpha-synuclein monomer precede the formation of Lewy bodies. We examined postmortem PD and undiseased (control) substantia nigra for evidence of pathological crosslinking of alpha-synuclein by tissue transglutaminase (tTG) using immunohistochemistry, immunoprecipitation, and Western blot. Consistent with previous reports, we found that both tTG and its substrate-characteristic N(epsilon)-(gamma-glutamyl)-lysine crosslink are increased in PD nigral dopamine neurons. Furthermore, both the tTG protein and its substrate crosslink coprecipitated with alpha-synuclein in extracts of PD substantia nigra. Unexpectedly, the isodipeptide crosslink was detected in the alpha-synuclein monomer as well as in higher molecular mass oligomers of alpha-synuclein. Although the intramolecularly crosslinked alpha-synuclein monomer was present in control tissue, it was highly enriched in PD substantia nigra. Conversely, significantly less uncrosslinked alpha-synuclein remained in the postimmunoprecipitate lysate of PD tissue than in control. Crosslinked alpha-synuclein, formed at the expense of the total alpha-synuclein monomer, correlated with disease progression. These results demonstrate that much of the alpha-synuclein monomer in PD nigra is crosslinked by tTG and thus may be functionally impaired. This modification appears to be an early step in PD pathogenesis, preceding the aggregation of alpha-synuclein in Lewy bodies.

The lysis genes of the virulent Staphylococcus aureus bacteriophage Twort were cloned and their nucleotide sequences determined. The endolysin gene plyTW encodes a 53.3-kDa protein, whose catalytic site is located in the amino-terminal domain. An enzymatically active fragment (N-terminal 271 amino acids) was overexpressed in Escherichia coli and partially purified. The enzyme rapidly cleaves staphylococcal peptidoglycan, and was shown to act as N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). Significant sequence homology to the specific cell wall targeting domain of lysostaphin was observed in a 101-amino acid C-terminal overlap. However, we found that the large C-terminal portion (63%, 295 aa) of PlyTW is not required for staphylolytic activity. Located upstream of and overlapping plyTW by 35 bp in a different reading frame (+1), we identified holTW, which starts with a single TTG triplet. The gene specifies a 185-amino acid (20.5 kDa) holin protein, which features two potential hydrophobic, antiparallel transmembrane domains, and a highly charged, acidic C-terminus. HolTW is the largest class II holin described to date. It can substitute for the defective allele in phase lambda S' amber mutants, both in trans from an expression plasmid, and from within gt11::holTW. The proposed function is the formation of unspecific membrane lesions to promote access of the endolysin to the bacterial peptidoglycan.

Tissue transglutaminase (tTG) belongs to the family of transglutaminase enzymes that catalyze the posttranslational modification of proteins via Ca(2+)-dependent cross-linking reactions. The catalytic action of tTG results in the formation of an isopeptide bond that is of great physiological significance since it is highly resistant to proteolysis and denaturants. Although tTG-mediated cross-linking reactions have been implicated to play a role in diverse biological processes, the precise physiological function of the enzyme remains unclear. Recent data, however, suggest that the protein polymers resulting from tTG-catalyzed reactions may play a role in commitment of cells to undergo apoptosis. On the same token, tTG-mediated formation of insoluble protein aggregates may underlie the markers of numerous pathological conditions, such as the senile plaques in Alzheimer's disease and the Lewy bodies in Parkinson's disease. In addition to catalyzing Ca(2+)-dependent cross-linking reactions, tTG can also bind and hydrolyze guanosine triphosphate and adenosine triphosphate. By virtue of this ability, tTG has been identified as a novel G-protein that interacts and activates phospholipase C following stimulation of the alpha-adrenergic receptor. The ability of tTG to mediate signal transduction may contribute to its involvement in the regulation of cell cycle progression. The following review summarizes the important features of this multifunctional enzyme that have emerged as a result of recent work from different laboratories.

The retinoblastoma gene product (pRB) plays an important role in controlling both cell release from the G1 phase and apoptosis. We show here that in the early phases of apoptosis, pRB is posttranslationally modified by a tissue transglutaminase (tTG)-catalyzed reaction. In fact, by employing a novel haptenized lysis synthetic substrate which allows the isolation of glutaminyl-tTG substrates in vivo, we identified pRB as a potential tTG substrate in U937 cells undergoing apoptosis. In keeping with this finding, we showed that apoptosis of U937 cells is characterized by the rapid disappearance of the 105,000- to 110,000-molecular-weight pRB forms concomitantly with the appearance of a smear of immunoreactive products with a molecular weight of greater than 250,000. The shift in pRB molecular weight was reproduced by adding exogenous purified tTG to extracts obtained from viable U937 cells and was prevented by dansylcadaverine, a potent enzyme inhibitor. The effect of the pRB posttranslational modification during apoptosis was investigated by determining the E2F-1 levels and by isolating and characterizing pRB-null clones from U937 cells. Notably, the lack of pRB in these U937-derived clones renders these p53-null cells highly resistant to apoptosis induced by serum withdrawal, calphostin C, and ceramide. Taken together, these data suggest that tTG, acting on the pRB protein, might play an important role in the cell progression through the death program.

A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert, and the putative hyaluronidase coding sequence was subcloned into pBluescript SK as a 3.8-kb PstI-ClaI fragment (designated pJCP802). The complete nucleotide sequence of this insert was determined. The region included an open reading frame sufficient to encode a polypeptide with an M(r) of 107,751. An active hyaluronidase with an M(r) of approximately 89,000 was purified to homogeneity from E. coli DH5 alpha(pJCP802). N-terminal amino acid sequence analysis of the purified protein suggested that translation initiation was occurring primarily at a TTG codon within the major open reading frame. However, immunoblot analysis using antiserum raised against the purified 89-kDa hyaluronidase indicated that E. coli DH5 alpha(pJCP802) also expressed the 107-kDa form of the enzyme. This antiserum labelled a 107-kDa protein in partially purified hyaluronidase preparations from S. pneumoniae. The hyaluronidase activity in this pneumococcal extract was also neutralized by the antiserum.

Tuberculosis (TB) is still a major health problem, both as a single disease entity and as a cofactor in AIDS. The interaction between macrophage and Mycobacterium tuberculosis (MTB) is a critical step in the establishment of an early chronic infection. This study analyses the capacity of MTB to induce apoptosis in cells obtained by broncho-alveolar lavage (BAL) from patients with reactive pulmonary tuberculosis and from AIDS patients with disseminated pulmonary tuberculosis. Apoptosis was increased three-fold in BAL cells obtained from patients with pulmonary tuberculosis and even more markedly in alveolar macrophages of MTB-infected AIDS patients, compared with controls. Apoptosis was analysed and characterized by propidium iodide (PI) incorporation, terminal deoxy transferase (TDT)-mediated dUTP-biotin nick end labelling (TUNEL), and tissue transglutaminase (tTG) expression. The MTB-macrophage interaction was also investigated in vitro by infecting monocyte-derived macrophages (MDM) with MTB (virulent strain H37Rv). The induction of apoptosis by MTB required viable bacteria, was dose-dependent, and was restricted to H37Rv. Infection with either Mycobacterium avium complex (MAC) or HIV-1 and treatment with heat-killed MTB failed to induce apoptosis.

A position-dependent pattern of epidermal cell types is produced during the development of the Arabidopsis seedling root and hypocotyl. To understand the origin and regulation of this patterning mechanism, we have examined the embryonic expression of the GLABRA2 (GL2) gene, which encodes a cell-type-specific transcription factor. Using in situ RNA hybridization and a sensitive GL2::GFP reporter, we discovered that a position-dependent pattern of GL2 expression is established within protodermal cells at the heart stage and is maintained throughout the remainder of embryogenesis. In addition, we show that an exceptional GL2 expression character and epidermal cell pattern arises during development of the root-hypocotyl junction, which represents an anatomical transition zone. Furthermore, we find that two of the genes regulating seedling epidermal patterning, TRANSPARENT TESTA GLABRA (TTG) and WEREWOLF (WER), also control the embryonic GL2 pattern, whereas the CAPRICE (CPC) and GL2 genes are not required to establish this pattern. These results indicate that position-dependent patterning of epidermal cell types begins at an early stage of embryogenesis, before formation of the apical meristems and shortly after the cellular anatomy of the protoderm and outer ground tissue layer is established. Thus, epidermal cell specification in the Arabidopsis seedling relies on the embryonic establishment of a patterning mechanism that is perpetuated postembryonically.

DQ2 confers susceptibility to celiac disease (CD) and intestinal CD4(+) T cells of DQ2(+) CD patients preferentially recognize deamidated gliadin peptides. This modification can be mediated by tissue transglutaminase (tTG). We have investigated what role the tTG-modified residues play in DQ2 binding and T cell presentation using a model gamma-gliadin peptide (residues 134 - 153). Treatment of this peptide with tTG resulted in deamidation of Gln residues at positions 140, 148 and 150. Two of these residues act as DQ2 anchors at position P7 (148) and P9 (150) and increased the affinity of the modified peptide for DQ2 50-fold. Testing of a mutant DQ2 molecule demonstrated that the Lys residue at beta71 of DQ2 is important for binding of the deamidated peptide. A variant DQ2 molecule (with the same beta-chain but different alpha-chain) that does not confer susceptibility to CD was capable of presenting the gliadin peptide, but not pepsin/trypsin-digested gliadin, equally well to a T cell. This suggests that processing events might be involved in the preferential presentation of the gliadin peptide by the DQ2 molecule. Substitution of Gln with Glu in some positions not targeted by tTG, but in positions likely to be deamidated via non-enzymatic mechanisms, disrupted T cell recognition. This provides additional evidence that tTG is responsible for modification of gliadin in vivo.

Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser->>Leu) and 11 (29.7%) had GAC in position 526 (His->>Asp).

BACKGROUND

Enterocyte damage is the hallmark of coeliac disease (CD) resulting in malabsorption. Little is known about the recovery of enterocyte damage and its clinical consequences. Serum intestinal fatty acid binding protein (I-FABP) is a sensitive marker to study enterocyte damage.

OBJECTIVE

To evaluate the severity of enterocyte damage in adult-onset CD and its course upon a gluten-free diet (GFD). Furthermore, the correlation among enterocyte damage, CD autoantibodies and histological abnormalities during the course of disease is studied.

METHODS

Serum I-FABP levels were determined in 96 biopsy-proven adult CD patients and in 69 patients repeatedly upon a GFD. A total of 141 individuals with normal antitissue transglutaminase antibody (IgA-tTG) levels served as controls. I-FABP levels were related to the degree of villous atrophy (Marsh grade) and IgA-tTG.

RESULTS

I-FABP levels were elevated in untreated CD (median 691 pg/mL) compared with controls (median 178 pg/mL, P < 0.001) and correlated with Marsh grade (r = 0.265, P < 0.05) and IgA-tTG (r = 0.403, P < 0.01). Upon a GFD serum levels decreased significantly, however, not within the range observed in controls, despite the common observed normalisation of IgA-tTG levels and Marsh grade. CD patients with elevated I-FABP levels nonresponding to GFD showed persistent histological abnormalities.

CONCLUSIONS

Enterocyte damage assessed by serum I-FABP correlates with the severity of villous atrophy in coeliac disease at the time of diagnosis. Although enterocyte damage improves upon treatment, substantial enterocyte damage persists despite absence of villous atrophy and low IgA-tTG levels in the majority of cases. Elevated I-FABP levels nonresponding to gluten-free diet are indicative of histological abnormalities and warrant further evaluation.