Systemic and local CC chemokines production in a murine model of Listeria monocytogenes infection

Repeated intragastric inoculation of Listeria monocytogenes into BALB/c mice resulted in prolonged bacteraemia and severe hepatic infection. Bacteria could also be isolated from the brain tissue of all experimental mice. During the inflammatory process, chemokine concentrations typically increased at the local site in comparison to the systemic level. The liver-to-serum ratio was more pronounced in the case of macrophage inflammatory protein 1 alpha (MIP-1 alpha), suggesting its role in the inflammatory response in the liver. The ratio of brain-to-serum concentration of monocyte chemoattractant protein 1 (MCP-1) remained the same as in the control animals, while it was lower in the infected mice, both in the case MIP-1alpha and in the case of regulated on activation, normal T cell expressed and secreted (RANTES). This is in correlation with slight inflammatory infiltrates found in the brain tissue early in infection.     

Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis

Lipid peroxidation product acrolein, well-known pollutant in tobacco and automotive smoke, accumulates in vivo bound to proteins. It suppresses p53 synthesis acting as potent carcinogenic factor for oral, respiratory and bladder carcinomas, while its possible association with colon carcinogenesis was not studied so far. We used genuine monoclonal antibody to evaluate immunohistochemical distribution of acrolein-protein adducts in 113 human colon tumours. The presence of acrolein-protein adducts was increasing with respect to colon carcinogenesis, from moderate appearance in tubular and villotubular low-grade adenomas to abundant and diffuse distribution in high-grade villotubular adenomas and Dukes A carcinomas. However, in advanced Dukes B and C carcinomas acrolein was hardly noticed, although, its protein adducts were found abundant in non-malignant colon epithelium of these patients. There was no relationship between p53 and acrolein distribution. According to these findings, acrolein seems to be lipid peroxidation product associated with transition from benign into malignant colon tumours.     

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.     

Structural determinants critical for localization and signaling within the seventh transmembrane domain of the type 1 corticotropin releasing hormone receptor: lessons from the receptor variant R1d

The type 1 CRH receptor (CRH-R1) plays a fundamental role in homeostatic adaptation to stressful stimuli. CRH-R1 gene activity is regulated through alternative splicing and generation of various CRH-R1 mRNA variants. One such variant is the CRH-R1d, which has 14 amino acids missing from the putative seventh transmembrane domain due to exon 13 deletion, a splicing event common to other members of the B1 family of G protein-coupled receptors. In this study, using overexpression of recombinant receptors in human embryonic kidney 293 and myometrial cells, we showed by confocal microscopy that in contrast to CRH-R1alpha, the R1d variant is primarily retained in the cytoplasm, although some cell membrane expression is also evident. Use of antibodies against the CRH-R1 C terminus in nonpermeabilized cells showed that membrane-expressed CRH-R1d contains an extracellular C terminus. Interestingly, treatment of CRH-R1d-expressing cells with CRH (100 nM) for 45-60 min elicited functional responses associated with a significant reduction of plasma membrane receptor expression, redistribution of intracellular receptors, and increased receptor degradation. Site-directed mutagenesis studies identified the cassette G356-F358 within transmembrane domain 7 as crucial for CRH-R1alpha stability to the plasma membrane because deletion of this cassette caused substantial intracellular localization of CRH-R1 alpha. Most importantly, coexpression studies between CRH-R1d and CRH-R2beta demonstrated that the CRH-R2beta could partially rescue CRH-R1d membrane expression, and this was associated with a significant attenuation of urocotrin II-induced cAMP production and ERK1/2 and p38MAPK activation, suggesting that CRH-R1d might specifically induce heterologous impairment of CRH-R2 signaling responses. This mechanism appears to involve accelerated CRH-R2beta endocytosis.     

Nuclear morphometry and AgNOR quantification: computerized image analysis on ovarian mucinous tumor imprints

OBJECTIVE: To determine the morphometric characteristics of nuclei and silver-stained nucleolar organizer regions (AgNORs) on cytologic imprints and their value in differential cytodiagnosis of benign, atypical proliferative (borderline) and malignant ovarian mucinous tumors. STUDY DESIGN: Forty-six mucinous ovarian tumor imprints (16 benign, 15 borderline, 15 malignant), were analyzed. Nuclear area, outline, "shape factor" and "form factor" were measured on Papanicolaou-stained smears. AgNOR quantification included 7 variables related to the number and area of single, cluster, total and relative AgNOR content per nucleus and the size distribution of AgNORs. RESULTS: Nuclear area and shape factor allowed distinguishing borderline and malignant tumors. The nuclear area in benign tumors was larger than that in borderline tumors; malignant tumors had the highest values. Single and cluster AgNORs were statistically significantly different in borderline tumors compared with malignant tumors, except for the cluster AgNOR area. The total AgNOR area, number and relative area increased from benign through malignant tumors, with statistically significant differences among all groups. By AgNOR size distribution, small AgNORs discriminate malignant from borderline and benign tumors. CONCLUSION: Combining nuclear morphometry and AgNOR analysis on cytologic imprints could be a diagnostically useful method in the assessment of mucinous ovarian tumors.     

Mutations that modulate receptor-hormone congruency as a cause of the primate GH receptor species specificity

Peptide hormones depend on reliable recognition by their receptors. Any mutation that compromises recognition of hormone and receptor molecules is dangerous, the carrier animal would not procreate and the mutation would be lost. Although, most of the hormones from one mammalian species are active when injected into another, the incompatibility of human GH receptor toward nonprimate GHs is a notable exception. It is reported that the coevolution of GH and GHR in primates includes two crucial steps (Mol. Biol. Evol. 18 (2001) 945). The first was mutation of GH His→Asp at position 171 that happened before the split of Old world and New world monkeys. The second event was Leu→Arg change at position 43 in the GH receptor molecule that happened in the ancestor of Old world monkeys. The proposed model is based on the possibility that certain mutations can modify the surface of one of interacting molecules to form a confined empty space, a niche in the otherwise congruent hormone/receptor interface. Although affinity between molecules is probably slightly reduced, recognition and function are not compromised in this special case. Further mutations of hormone and receptor molecules are allowed under the condition that they remain confined to the niche space. Mutations that do not compromise hormone function can be passed to offsprings. If the consequent mutation of one molecule change its shape to fill the niche space, further mutations without function loss will become less probable. Without the niche space, the phase of fast evolution is closed and both genes become conserved. In this setting, accumulated mutations before the niche closing mutation are the cause of species specificity. To become a dominant variety, carrier animals must possess survival advantage in comparison to the carriers of other less advantageous mutations.     

Risk of pneumonia recurrence in patients previously hospitalized for pneumonia--a retrospective study (1998-2000)

Although elderly hospitalized patients, irrespective of the cause of hospitalization, are known to be at a high risk of subsequent development of pneumonia, some studies suggest the risk to be even higher in those hospitalized for pneumonia than in those hospitalized for other diseases. The aim of this retrospective study was to determine the association of hospitalization for pneumonia and some other diseases with subsequent pneumonia morbidity and mortality. The risk of recurrent pneumonia in patients hospitalized for pneumonia was investigated. Rehospitalization of pneumonia patients previously hospitalized for the same disease was followed-up and compared with rehospitalization of patients hospitalized for other diseases during the same study period. The study included patients aged overl8, initially hospitalized in 1998 for pneumonia (J12-J18), or for some particular gastrointestinal (K20-K31) and urogenital diseases (N10-N12, N30-N39). All rehospitalizations for pneumonia in nine Zagreb hospitals were followed-up during a 3-year study period (1998-2000). Out of 975 patients followed-up for rehospitalization, 227 (23.3%) had initially been hospitalized for pneumonia, and 748 (76.7%) for other diagnoses. During the 3-year period, 30 patients were rehospitalized for pneumonia, out of which number 22 had initially been hospitalized for pneumonia, yielding a statistically significant difference between the two study groups (chi2 = 34.780, p < 0.001). The mortality directly caused by pneumonia was also significantly higher in the group of patients with the initial diagnosis of pneumonia than in the group of patients with other diagnoses (chi2 = 15.82, p < 0.001).     

Impaired transporter associated with antigen processing-dependent peptide transport during productive EBV infection

Human herpesviruses, including EBV, persist for life in infected individuals. During the lytic replicative cycle that is required for the production of infectious virus and transmission to another host, many viral Ags are expressed. Especially at this stage, immune evasion strategies are likely to be advantageous to avoid elimination of virus-producing cells. However, little is known about immune escape during productive EBV infection because no fully permissive infection model is available. In this study, we have developed a novel strategy to isolate populations of cells in an EBV lytic cycle based on the expression of a reporter gene under the control of an EBV early lytic cycle promoter. Thus, induction of the viral lytic cycle in transfected EBV(+) B lymphoma cells resulted in concomitant reporter expression, allowing us, for the first time, to isolate highly purified cell populations in lytic cycle for biochemical and functional studies. Compared with latently infected B cells, cells supporting EBV lytic cycle displayed down-regulation of surface HLA class I, class II, and CD20, whereas expression levels of other surface markers remained unaffected. Moreover, during lytic cycle peptide transport into the endoplasmic reticulum, was reduced to <30% of levels found in latent infection. Because steady-state levels of TAP proteins were unaffected, these results point toward EBV-induced interference with TAP function as a specific mechanism contributing to the reduced levels of cell surface HLA class I. Our data implicate that EBV lytic cycle genes encode functions to evade T cell recognition, thereby creating a window for the generation of viral progeny.