Glucuronidation of the mycotoxins alternariol and alternariol-9-methyl ether in vitro: chemical structures of glucuronides and activities of human UDP-glucuronosyltransferase isoforms.

Alternariol (AOH) and alternariol-9-methyl ether (AME) are major toxins produced by fungi of the genus Alternaria and are frequently found in various food items. Because AOH has three hydroxyl groups and AME two, the formation of various glucuronides must be expected. When AOH was incubated with hepatic and intestinal microsomes from rats, pigs and humans in the presence of uridine diphosphate glucuronic acid, two glucuronides were detected and tentatively identified as AOH-3-O-glucuronide and AOH-9-O-glucuronide. Under the same conditions, AME yielded predominantly AME-3-O-glucuronide and only small amounts of AME-7-O-glucuronide. The activities of all microsomes for the glucuronidation of AOH and AME were in the same range. Nine out of ten recombinant human UDP-glucuronosyltransferases (UGTs) were able to glucuronidate AOH, and eight out of ten UGTs had activity for AME. These data suggest that AOH and AME are readily glucuronidated in hepatic and extrahepatic tissues, implying that glucuronidation constitutes a major metabolic pathway in the disposition of these mycotoxins.

Activities of human recombinant cytochrome P450 isoforms and human hepatic microsomes for the hydroxylation ofAlternaria toxins.

TheAlternaria toxins alternariol (AOH), alternariol-9-methyl ether (AME), altenuene (ALT) and isoaltenuene (iALT) undergo extensive oxidative metabolism, but the cytochrome P450 (CYP) isoforms responsible for the reported hydroxylation reactions are yet unknown. In the present study, the activities of twelve human CYP isoforms for the hydroxylation of AOH, AME, ALT and iALT at different positions have been determined. The most active monooxygenase for AOH and AME was CYP1A1, and lower activities were observed for CYP1A2, 2C19 and 3A4. Hydroxylation at C-2 of AOH and AME was the preferred reaction of most isoforms. For ALT and iALT, CYP2C19 had the highest activity, followed by 2C9 and 2D6. The dominating metabolite of all active isoforms was the 8-hydroxylated ALT and iALT. The activities of the CYP isoforms are consistent with the pattern of metabolites of theAlternaria toxins obtained with pooled human hepatic microsomes. Based on the activities of the CYP isoforms, a significant extrahepatic hydroxylation must be expectede.g. in the lung and esophagus for AOH and AME, and in the intestine and ovaries for ALT and iALT. As all major hydroxylation products are catechols, the extrahepatic metabolism ofAlternaria toxins may be of toxicological relevance.

Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65).

CD8 T cells are the principal antiviral effectors controlling cytomegalovirus (CMV) infection. For human CMV, the virion tegument protein ppUL83 (pp65) has been identified as a source of immunodominant peptides and is regarded as a candidate for cytoimmunotherapy and vaccination. Two sequence homologs of ppUL83 are known for murine CMV, namely the virion protein ppM83 (pp105) expressed late in the viral replication cycle and the nonstructural protein pM84 (p65) expressed in the early phase. Here we show that ppM83, unlike ppUL83, is not delivered into the antigen presentation pathway after virus penetration before or in absence of viral gene expression, while other virion proteins of murine CMV are processed along this route. In cytokine secretion-based assays, ppM83 and pM84 appeared to barely contribute to the acute immune response and to immunological memory. Specifically, the frequencies of M83 and M84 peptide-specific CD8 T cells were low and undetectable, respectively. Nonetheless, in a murine model of cytoimmunotherapy of lethal CMV disease, M83 and M84 peptide-specific cytolytic T-cell lines proved to be highly efficient in resolving productive infection in multiple organs of cell transfer recipients. These findings demonstrate that proteins which fail to prime a quantitatively dominant immune response can nevertheless represent relevant antigens in the effector phase. We conclude that quantitative and qualitative immunodominance are not necessarily correlated. As a consequence of these findings, there is no longer a rationale for considering T-cell abundance as the key criterion for choosing specificities to be included in immunotherapy and immunoprophylaxis of CMV disease and of viral infections in general.

Synthesis of beta-lactams from diazoketones and imines: the use of microwave irradiation.

[see reaction]. The transformation of diazoketones derived from alpha-amino acids to ketenes that, in turn, react further with imines to afford beta-lactams, can be realized not only by utilizing photochemical reaction conditions but also under the action of microwave irradiation. Under the latter reaction conditions 4-alkenyl-substituted beta-lactams derived from amino acids, substrates that were not previously accessible, have been prepared. beta-Lactams possessing a trans-substitution pattern at the ring were obtained exclusively.

Origin of organic molecules and biomolecular homochirality.

Theories about the origin of biomolecular homochirality, which seems to be a prerequisite for the creation of life, are discussed. First, possible terrestrial and extraterrestrial sources of organic molecules are outlined. Then, mechanisms for the formation of enantiomerically enriched compounds and for the amplification of their chirality are described.

Murine model of interstitial cytomegalovirus pneumonia in syngeneic bone marrow transplantation: persistence of protective pulmonary CD8-T-cell infiltrates after clearance of acute infection.

Interstitial pneumonia (IP) is a severe organ manifestation of cytomegalovirus (CMV) disease in the immunocompromised host, in particular in recipients of bone marrow transplantation (BMT). Diagnostic criteria for the definition of CMV-IP include clinical evidence of pneumonia together with CMV detected in bronchoalveolar lavage or lung biopsy. We have used the model of syngeneic BMT and simultaneous infection of BALB/c mice with murine CMV for studying the pathogenesis of CMV-IP by controlled longitudinal analysis. A disseminated cytopathic infection of the lungs with fatal outcome was observed only when reconstituting CD8 T cells were depleted. Neither CD8 nor CD4 T cells mediated an immunopathogenesis of acute CMV-IP. By contrast, after efficient hematolymphopoietic reconstitution, viral replication in the lungs was moderate and focal. The histopathological picture was dominated by preferential infiltration of CD8 T cells confining viral replication to inflammatory foci. Notably, after clearance of acute infection, CD62L(lo) and CD62L(hi) subsets of CD44(+) memory CD8 T cells were found to persist in lung tissue. One can thus operationally distinguish an early CMV-positive IP (phase 1) and a late CMV-negative IP (phase 2). According to the definition, phase 2 histopathology would not be diagnosed as a CMV-IP and could instead be misinterpreted as a CMV-induced immunopathology. We document here that phase 1 as well as phase 2 pulmonary CD8 T cells are capable of exerting effector functions and are effectual in protecting against productive infection. We propose that antiviral "stand-by" memory-effector T cells persist in the lungs to prevent virus recurrence from latency.

The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells.

Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the "missing self." The retention, however, is counteracted by the m04 early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule D(d). Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1(168-176)), the early nonapeptide m04(243-251) is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.

In vivo replication of recombinant murine cytomegalovirus driven by the paralogous major immediate-early promoter-enhancer of human cytomegalovirus.

Transcription of the major immediate-early (MIE) genes of cytomegaloviruses (CMV) is driven by a strong promoter-enhancer (MIEPE) complex. Transactivator proteins encoded by these MIE genes are essential for productive infection. Accordingly, the MIEPE is a crucial control point, and its regulation by activators and repressors is pertinent to virus replication. Since the MIEPE contains multiple regulatory elements, it was reasonable to assume that specific sequence motifs are irreplaceable for specifying the cell-type tropism and replication pattern. Recent work on murine CMV infectivity (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502-8509, 1998) has documented the proposed enhancing function of the enhancer in that its resection or its replacement by a nonregulatory stuffer sequence resulted in a significant reduction of infectivity, even though replication competence was maintained by a basal activity of the spared authentic MIE promoter. Notably, full capacity for productive in vitro infection of fibroblasts was restored in recombinant viruses by the human CMV enhancer. Using two-color in situ hybridization with MIEPE-specific polynucleotide probes, we demonstrated that a murine CMV recombinant in which the complete murine CMV MIEPE is replaced by the paralogous human CMV core promoter and enhancer (recombinant virus mCMVhMIEPE) retained the potential to replicate in vivo in all tissues relevant to CMV disease. Notably, mCMVhMIEPE was also found to replicate in the liver, a site at which transgenic hCMV MIEPE is silenced. We conclude that productive in vivo infection with murine CMV does not strictly depend on a MIEPE type-specific regulation.

Apoptosis mediated by Fas but not tumor necrosis factor receptor 1 prevents chronic disease in mice infected with murine cytomegalovirus.

The role of Fas- and TNF-receptor 1 (TNF-R1)-mediated apoptosis in the clearance of virally infected cells and in the regulation of the immune response was analyzed after murine cytomegalovirus (MCMV) infection of C57BL/6 (B6)-+/+ mice, Fas-mutant B6-lpr/lpr mice, TNF-R1 knockout B6-tnfr0/0 mice, and double-deficient B6-tnfr0/0 lpr/lpr mice. There was approximately equivalent clearance of MCMV in B6-+/+, B6-tnfr0/0, and B6-lpr/lpr mice, and by day 28 no infectious virus could be detected in the liver, kidney, lung, or peritoneal exudate. However, delayed virus clearance was observed in B6-tnfr0/0 lpr/lpr mice. An acute inflammatory response occurred in the liver, lung, and kidney of all mice, which was most severe 7 d after MCMV infection, but resolved by day 28 in B6-+/+ and B6-tnfr0/0 mice, but not in B6-lpr/lpr or B6-tnfr0/0 lpr/lpr mice. These results indicate that apoptosis mediated by either Fas or TNF-R1 is sufficient for rapid clearance of the virus. However, apoptosis induced by Fas, but not TNF-R1, is required for the downmodulation of the immune response to the virus and prevention of a chronic inflammatory reaction.

Reconstitution of CD8 T cells is essential for the prevention of multiple-organ cytomegalovirus histopathology after bone marrow transplantation.

Cytomegalovirus (CMV) infection in the period of temporary immunodeficiency after haematoablative treatment and bone marrow transplantation (BMT) is associated with a risk of graft failure and multiple-organ CMV disease. The efficacy of immune system reconstitution is decisive for the prevention of CMV pathogenesis after BMT. Previous data in murine model systems have documented a redundancy in the immune effector mechanisms controlling CMV. CD8 T cells proved to be relevant but not irreplaceable as antiviral effectors. Specifically, in a state of long-term in vivo depletion of the CD8 T-cell subset, CD4 T cells were educed to become deputy effectors controlling CMV by a mechanism involving antiviral cytokines. It is of medical importance to know whether one can trust in this 'flexible defence' in all clinical settings. It is demonstrated here that reconstitution of CD8 T cells is crucial for the prevention of fatal multiple-organ CMV disease under the specific conditions of BMT.

Control of cytomegalovirus in bone marrow transplantation chimeras lacking the prevailing antigen-presenting molecule in recipient tissues rests primarily on recipient-derived CD8 T cells.

Cytomegalovirus (CMV) infection during the transient immunodeficiency after bone marrow transplantation (BMT) develops into disease unless antiviral CD8 T cells are restored in due course. Histoincompatibility between donor and recipient is associated with increased risk. Complications may include a rejection response against the foreign major histocompatibility complex (MHC) antigens and a lack of antiviral control resulting from a misfit between donor-derived T cells and the antigenic viral peptides presented in recipient tissues. Here we have established a murine model of CMV disease after experimental BMT performed across a single MHC class I disparity. Specifically, BALB/c bone marrow cells expressing the prevailing antigen-presenting molecule Ld were transplanted into the Ld gene deletion mutant BALB/c-H-2(dm2), an experimental setting that entails a selective risk of host-versus-graft but not graft-versus-host response. The reconstituted T-cell population proved to be chimeric in that it consisted of Ld-positive donor-derived and Ld-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against Ld. This finding implies that the infection did not trigger a host-versus-graft response. Notably, upon adoptive transfer, donor-derived CD8 T cells preferentially protected tissues of donor genotype, whereas recipient-derived CD8 T cells protected tissues of either genotype. We infer from these data that the focus on immunodominant antigens presented by Ld within the donor cell population distracted the donor T cells from protecting recipient tissues and that protection in the chimeras was therefore primarily based on recipient T cells. As a consequence, T-cell chimerism after BMT should give a positive prognosis with respect to control of CMV.

Control of murine cytomegalovirus in the lungs: relative but not absolute immunodominance of the immediate-early 1 nonapeptide during the antiviral cytolytic T-lymphocyte response in pulmonary infiltrates.

The lungs are a major organ site of cytomegalovirus (CMV) infection, pathogenesis, and latency. Interstitial CMV pneumonia represents a critical manifestation of CMV disease, in particular in recipients of bone marrow transplantation (BMT). We have employed a murine model for studying the immune response to CMV in the lungs in the specific scenario of immune reconstitution after syngeneic BMT. Control of pulmonary infection was associated with a vigorous infiltration of the lungs, which was characterized by a preferential recruitment and massive expansion of the CD8 subset of alpha/beta T cells. The infiltrate provided a microenvironment in which the CD8 T cells differentiated into mature effector cells, that is, into functionally active cytolytic T lymphocytes (CTL). This gave us the opportunity for an ex vivo testing of the antigen specificities of CTL present at a relevant organ site of viral pathogenesis. The contribution of the previously identified immediate-early 1 (IE1) nonapeptide of murine CMV was evaluated by comparison with the CD3epsilon-redirected cytolytic activity used as a measure of the overall CTL response in the lungs. The IE1 peptide was detected by pulmonary CTL, but it accounted for a minor part of the response. Interestingly, no additional viral or virus-induced antigenic peptides were detectable among naturally processed peptides derived from infected lungs, even though infected fibroblasts were recognized in a major histocompatibility complex-restricted manner. We conclude that the antiviral pulmonary immune response is a collaborative function that involves many antigenic peptides, among which the IE1 peptide is immunodominant in a relative sense.

The human autoantigen La/SS-B accelerates herpes simplex virus type 1 replication in transfected mouse 3T3 cells.

Permanently transfected mouse cell lines which expressed different levels of the human autoantigen La/SS-B were infected with different strains of herpes simplex virus type 1, including the strains ANG, HSZP, 17syn+ and HFEM. During infection the localization of the human La protein was followed using an anti-La MoAb, which recognized only the human La protein but did not cross-react with either the endogenous mouse La protein or any viral encoded protein. After infection La protein was transported from the nucleus to the cytoplasm. The time course of translocation was dependent on the amount of human La protein expressed in the respective cell line. Moreover, acceleration of viral replication was dependent on the level of expression of human La protein, suggesting that La protein is a cellular factor that facilitates virus replication.

Cytomegalovirus inhibits the engraftment of donor bone marrow cells by downregulation of hemopoietin gene expression in recipient stroma.

Cytomegalovirus (CMV) disease after bone marrow (BM) transplantation is often associated with BM graft failure. There are two possible reasons for such a correlation. First, a poor hematopoietic reconstitution of unrelated etiology could promote the progression of CMV infection by the lack of immune control. Alternatively, CMV infection could interfere with the engraftment of donor BM cells in recipient BM stroma. Evidence for a causative role of CMV in BM aplasia came from studies in long-term BM cultures and from the murine in vivo model of CMV-induced aplastic anemia. A deficiency in the expression of essential stromal hemopoietins, such as stem cell factor (SCF), has indicated a functional insufficiency of the stromal microenvironment. It remained open to question whether CMV mediates a negative regulation of hemopoietin gene expression (the downregulation model) or whether it causes the default of a positive regulator (the lack-of-induction model). Further, even though implicitly assumed, it has never been formally documented that CMV directly interferes with the engraftment of a BM cell transplant. We addressed these problems in a murine model of CMV infection after experimental male-into-female BM transplantation. The data indicate that the downregulation model applies. Quantitation of the male-sex-determining gene tdy demonstrated an impaired engraftment of donor BM cells in the BM stroma of the female recipients. This graft failure was reflected by a diminished population of SCF-receptor-expressing hematopoietic progenitor cells and correlated with a reduced level of stromal SCF gene expression. Interestingly, high doses of BM cells protected against stromal insufficiency by a mechanism unrelated to control of infection.

Evidence against a key role for transforming growth factor-beta1 in cytomegalovirus-induced bone marrow aplasia.

During immunodeficiency after sublethal haematoablative treatment, cytomegalovirus (CMV) infection interferes with haematopoietic reconstitution and can cause lethal bone marrow (BM) aplasia. The in vivo model of murine CMV infection has identified the BM stroma as the principal target site of CMV in the haematopoietic cord. The infected cell type is the reticular stromal cell which forms the stromal network and produces essential haemopoietins, such as stem-cell factor (SCF). The expression of SCF was found to be reduced in the infected stroma, but the stromal network was not disrupted and the number of infected stromal cells was too low to explain the functional deficiency. These facts call for a negatively regulating cytokine that is induced by the infection and that potentiates the direct effect of infection by down-regulating haemopoietins in uninfected bystander cells. Recent work has suggested that transforming growth factor (TGF)-beta1 might be the cytokine involved in CMV-induced BM aplasia. We show here that murine CMV indirectly induces the accumulation of mature TGF-beta1 in uninfected renal tubular epithelial cells and TGF-beta1 transcription in BM stromal cells, whereas infected renal glomerular and interstitial cells, hepatocytes and BM stromal cells do not coexpress mature TGF-beta1. Antiviral CD8 T-cell therapy prevented BM aplasia and also prevented the down-regulation of stromal SCF and interleukin-6 gene expression. Interestingly, however, the CD8 T cells did not preclude the up-regulation of mature TGF-beta1, but proved to be inducers of TGF-beta1 gene expression in BM stroma. These findings suggest that TGF-beta1 is not the mediator of BM aplasia.

Bone marrow failure by cytomegalovirus is associated with an in vivo deficiency in the expression of essential stromal hemopoietin genes.

Bone marrow (BM) failure associated with cytomegalovirus (CMV) infection is a feared complication after clinical BM transplantation. Experiments in long-term BM cultures have indicated that BM stromal cells (BMSC) are targets of productive CMV infection, but an in situ infection of BM stroma remained to be documented, and the pathomechanism is open to question. Here we describe a murine in vivo model of lethal CMV aplastic anemia (CMV-AA). The reconstitution of hematopoietic progenitor cells expressing stem cell factor (SCF) receptor was found to be defective in CMV-AA. While murine CMV replication in permissive parenchymal tissues is cytolytic, the hematopoietic cord was found to be a site of very limited virus production with foci of reticular BMSC expressing the intranuclear viral IE1 protein, but with only a few BMSC positive for viral genome in the in situ hybridization. XX-XY BM chimeras were established in order to quantitate Y-chromosome-tagged BMSC by a PCR specific for the male-sex-determining gene Tdy. This approach revealed that murine CMV infection is not associated with a significant loss of BMSC. Despite the physical integrity of the stromal network, the functional integrity of the stroma was impaired. While housekeeping genes were expressed normally in BMSC of infected mice, the expression of genes encoding the essential hemopoietins SCF, granulocyte colony-stimulating factor, and interleukin-6 was markedly reduced. In conclusion, the mechanism of BM failure is not a stromal lesion but an insufficient stromal function. These findings explain CMV-AA as a manifestation of multiple hemopoietin deficiency.

Localization of latency-associated transcripts in the uterovaginal plexus of herpes simplex virus type 1 and 2 latently infected mice.

The vagina and medulla of the adrenal gland of mice vaginally infected with herpes simplex virus (HSV) types 1 and 2 were examined in the latent stage of infection (5 to 51 weeks post-infection). RNA in situ hybridization with HSV-1 and -2 latency-associated transcript (LAT) RNA probes resulted in positively stained neuronal cell nuclei in the uterovaginal plexus, but not in the medulla of the adrenal gland. These organs were chosen because HSV antigens can be detected not only in the vaginal epithelium, but also in neurons of the uterovaginal plexus and in the medulla of the adrenal gland at the acute stage of genital infection. To our knowledge, this is the first report describing LATs in neurons of the uterovaginal plexus in the genital tract of latently HSV-infected mice.

Replication of herpes simplex virus type 1 and 2 in the medulla of the adrenal gland after vaginal infection of mice.

After vaginal infections of mice with neuroinvasive strains of herpes simplex virus type 1 and 2 (HSV-1, HSV-2) virus replicates in the epithelium of the vagina, in the paravaginal ganglia, in the spinal cord and finally in the brain and in the adrenal glands. However, viral antigens could be demonstrated only in the medulla of the adrenal glands but not in the cortex, as assessed by immunohistochemistry (IHC). HSV could not be isolated from liver, spleen, uterus, and ovaries. This contrasts to the intraperitoneal (i.p) route of infection with replication in different visceral organs including the adrenal gland's cortex.

Asymptomatic vaginal herpes simplex virus infections in mice: virology and pathohistology.

One of the causes of genital tract infections in humans are herpes simplex virus types 1 and 2 (HSV-1, HSV-2). Although primary and recurrent infections can be clinically apparent and in part very serious, many infections are asymptomatic and result only in temporary genital shedding of virus (recurrences). During our investigations of vaginitis, strain IES of HSV-1 produced an asymptomatic infection. Replication in the murine vaginal (vag.) epithelium as well as antibody formation after vag. infection was comparable to those of survivors after infection with highly virulent strains. Titration of liver, spleen, ovaries, adrenal glands spinal cord, or brain after vag. IES infection revealed no virus, whereas after i.p. infection virus could be demonstrated in many organs examined. Histological examination with a DNA probe (in situ hybridisation), HSV antibodies (immunohistochemistry), and haematoxylin and eosin (HE) staining showed only small focal HSV lesions of the vaginal epithelium in early stages of the infection, never exceeding to the subepithelial tissue. Severe infiltrations and ulcerations after infection with highly virulent strains (17syn +, ER-) could never be demonstrated after IES vag. infection. Identical replication rates of both groups of HSV despite much greater areas of epithelial necrosis with the virulent strains may be explained by the large number of virus inactivating granulocytes induced by the virulent strains, thus inactivating the hypothetical higher virus load.

Bilateral agenesis/aplasia of the lungs: report of a second case in the offspring of one woman.

Congenital absence of both lungs is an extremely rare malformation in humans and is thought to occur sporadically. We report the second case of congenital absence of both lungs in the offspring of one woman. In neither case, one female baby (born at term) and one aborted female fetus (21 weeks of gestation), were anomalies or malformations of other organ systems observed. The karyotype of the aborted fetus was 46,XX. To our knowledge, this is the first report describing bilateral pulmonary agenesis in two offspring of one mother. The repetition of virtually the same isolated abnormality with no other malformations supports the hypothesis that it could be caused by a genetic disorder. Other etiologies previously suggested, such as drugs or viruses, cannot be excluded but seem less likely.