Invasive zygomycosis in patients with graft-versus-host disease after allogeneic stem cell transplantation.

Invasive mold infections are a threat to immunosuppressed patients such as patients with graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). Up to 10% of SCT recipients develop invasive aspergillosis (IA). Invasive zygomycosis (IZ) may occur during treatment against IA. Here we report 4 SCT patients with GVHD diagnosed with IZ. All patients had received myeloablative hematopoietic SCT and developed chronic GVHD requiring systemic immunosuppression. Underlying diseases were acute lymphocytic leukemia (2), osteomyelofibrosis, and multiple myeloma. All patients had developed pulmonary infiltration that led to initiation of antifungal therapy. Treatment for IA was voriconazole, caspofungin, or itraconazole. Organs involved with zygomycosis were lung, nasal sinus, skin, and kidney. Treatment with liposomal amphotericin and posaconazole was initiated in all patients, and 2 patients also had surgical debridement as well. Despite intensive treatment, no patient survived. IZ is becoming more common in patients with GVHD on successful treatment for IA. Even non-specific symptoms are suspicious in this group of patients and need to be evaluated by vigorous diagnostics. Despite effective antifungals and surgical intervention, the prognosis is grim in patients with active GVHD, as immunoreconstitution is mandatory for successful management.

Subcutaneous fistulae in a patient with femoral hypoplasia due to Actinomyces europaeus and Actinomyces turicensis.

Infections due to Actinomyces europaeus or Actinomyces turicensis have only rarely been reported. We describe a case of chronic fistulae caused by a coinfection with A. europaeus and A. turicensis in an immunocompetent male patient with a severe congenital femur hypoplasia. Actinomycosis is most probably the consequence of a postoperative wound infection after a prior surgical intervention. Both Actinomyces species were identified by 16S rRNA gene sequencing. The Actinomyces-caused fistulae were treated by excision and a 1-week course of i.v. vancomycin followed by a 1-week course of p.o. cefuroxime.

PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue.

BACKGROUND: Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities. AIMS: To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples. METHODS: DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes. RESULTS: Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another two cases. CONCLUSIONS: The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment.

A rev/beta-galactosidase fusion protein binds in vitro transcripts spanning the rev-responsive element of human immunodeficiency virus type 1 (HIV-1).

The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular mass, is essential to target the mRNA for virion polypeptides into the cytoplasm. This effect is mediated by a specific RNA stretch (rev-responsive element = RRE) localized within a 3'-terminal segment of the mRNA encoding virion proteins. We present evidence that rev expressed as a beta-galactosidase fusion protein in E. coli forms a complex with in vitro transcripts containing the RRE; it can be precipitated by monoclonal antibodies with rev or beta-galactosidase specificity. In addition, specific binding of rev protein to RNA could be demonstrated by Northwestern blotting.

Molecular pathogenesis of Epstein-Barr virus associated posttransplant lymphomas: new insights through latent membrane protein 1 fingerprinting.

BACKGROUND: Fingerprint amino acid patterns within the carboxy terminus of the latent membrane protein (LMP1) oncoprotein of Epstein-Barr virus (EBV) allow individual strain identification at the molecular level. LMP1 is expressed in the tumor cells of EBV-associated posttransplant lymphomas (PTLs) and the LMP1 genome is also identified in lymphocytes of most donors of allogeneic bone marrow. Therefore, LMP1 genotyping in donor lymphocytes and PTL tumor cells, together with sex chromatin determination of tumor cells, allows to determine the origin of PTL tumor cells and the origin of individual EBV strains harboured by them. METHODS: We traced the origin of aggressive PTLs occurring in six patients after allogeneic T cell-depleted stem cell transplantation (allo-SCT). DNA was extracted from donor lymphocytes and PTLs of recipients and amplified with LMP1-specific primers in each case. A comparative sequence analysis of the fingerprint LMP1 region identified in donor lymphocytes and lymphoma was performed. RESULTS: One lymphoma of donor origin occurred after highly selected CD34+ PBSCT and contained the same LMP1 genotype as the donor lymphocytes. Three lymphomas of recipient origin had deletions within the carboxy terminus of LMP1, not identified in the donor strains. All lymphomas occurred in the setting of allo-SCT and had a rapid clinical course. CONCLUSIONS: These results show that highly selected CD34+ PBSCT does not protect against transfer of EBV positive founder cells of donor type PTL and that, after allo-SCT, recipient type PTLs are not uncommon. Outgrowth of recipient type lymphoma may be favoured by LMP1 deletion variant strains present in recipient lymphocytes.

Mechanisms and consequences of enterovirus persistence in cardiac myocytes and cells of the immune system.

In humans and experimental murine models enteroviruses, and in particular coxsackieviruses of group B (CVB), may induce chronic myocarditis associated with a persistent type of heart muscle infection. Persistent myocardial infection has been characterized by restricted viral replication and gene expression, which is capable of sustaining chronic inflammation. Altered replication and transcription of the virus, in addition to an immune response insufficient to recognize and clear infected cells entirely, are essential mechanisms for initiation and maintenance of persistent heart muscle infection. Viral cytotoxicity was found to be crucial for organ pathology both during acute and persistent infection, indicating that enterovirus myocarditis is a virus-induced rather than an immune-mediated disease. Notably, resistance to the development of persistent heart muscle infection is not linked to the H-2 haplotype of the host. In addition to persistently infected myocytes, detection of the replicative minus-strand RNA intermediate provided evidence for virus replication in lymphoid cells of the spleen, predominantly in splenic B lymphocytes, during the course of the disease. Whereas viral RNA was also detected in certain CD4+ helper T cells and Mac1+ macrophages, no enteroviral genomes were identified in CD8+ T cells. Detection of infected activated B lymphocytes both in heart tissue of CVB3-infected immunocompetent mice and syngenic SCID mice receiving splenocytes from CVB3-infected donors support the concept that B cell traffic may contribute to maintenance of chronic disease. Dissection of the diversity of viral and host-specific determinants in susceptible and resistant hosts will allow us to define the protective mechanisms that mediate resistance to the development of life-threatening acute and chronic enterovirus myocarditis.

Cationization of a monoclonal antibody to the human immunodeficiency virus REV protein enhances cellular uptake but does not impair antigen binding of the antibody.

Replication of the human immunodeficiency virus (HIV) within cells may be blocked by neutralization of viral-specific proteins that are absolutely required for growth of the virus. One such viral-specific protein is REV, and a monoclonal antibody (mAb) against the REV protein is a potential therapeutic for acquired immune deficiency syndrome (AIDS). However, in order to effect 'intracellular immunization', mAbs must be enabled to target the intracellular compartment. One strategy for transcellular drug delivery of mAb-based therapeutics is cationization, and the present studies describe the cationization of a murine mAb specific to the REV protein of HIV-1. The isoelectric point (pI) of the mAb was raised from 6.6 to more than 9.5. There was virtually no difference in binding to wild-type REV protein between the native or cationized anti-REV mAb, based on studies with a solid-phase immunoradiometric assay. The uptake of the [125I] native anti-REV mAb by human peripheral blood lymphocytes (PBLs) was negligible; however, there was a marked increase in both total cell binding and endocytosis by the human PBLs of the [125I] cationized anti-REV mAb. In conclusion, these studies show that an anti-REV mAb may be cationized to markedly increase endocytosis of the antibody and that this cationization reaction does not significantly alter the affinity of the antibody for its target protein. Cationized anti-REV mAbs may allow for intracellular immunization of the virus and are potential therapeutics for the treatment of HIV.

Extranodal marginal zone B cell lymphoma of MALT type involving the mucosa of both the urinary bladder and stomach.

Primary lymphoma of the urinary bladder is a very rare tumour. A bladder tumour was found in a 57 year old man with obstructive dysuria. It was found by histological and immunohistohistochemical investigation to be an extranodal marginal zone B cell lymphoma. Lymphoepithelial lesions were absent, but were found in a clinically silent gastric lymphoma discovered four weeks later during staging investigations; this gastric lymphoma was negative for Helicobacter pylori by breath test and molecular biological analysis. Sequencing of the clonal immunoglobulin heavy chain gene in both tumours indicated the same precursor cell, of follicular or post follicular origin. In synopsis, the data suggested that this was a case of primary lymphoma of the bladder with involvement of the stomach. The application of a chromosome 3 specific alpha satellite probe revealed trisomy 3. A tumour with these characteristics arising as a lymphoma of the bladder with a metachronous involvement of the gastric mucosa has not been described previously.

Activity of the antiseptic polyhexanide against meticillin-susceptible and meticillin-resistant Staphylococcus aureus.

Staphylococcus aureus is one of the most important pathogens, with increasing emergence of meticillin-resistant S. aureus (MRSA) strains. This is associated not only with multiresistance to antibiotics but also with increasing resistance to topical antibiotics and antiseptics. As the antiseptic polyhexanide has only a low risk of emergence of resistant strains, the aim of the study was to obtain data on the sensitivity of S. aureus towards polyhexanide. The effect of polyhexanide was tested against 80 meticillin-susceptible S. aureus (MSSA) and 80 MRSA strains from sporadic cases as well as against 6 MRSA outbreak strains. The clonal diversity of the 166 strains was proven by pulsed-field gel electrophoresis (PFGE). Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined by the serial broth microdilution technique according to DIN 58940. Time-kill studies were performed for reference strains MSSA ATCC 29213 and MRSA ATCC 33591. MICs and MBCs in the range of 0.5-2mg/L were found. According to a created epidemiological cut-off (ECOFF) value of 4mg/L, all strains were regarded as susceptible to polyhexanide, including MRSA epidemic strains and MSSA and MRSA sporadic strains with various antibiotic susceptibility patterns. Addition of up to 4% albumin to the test medium did not change the MICs and MBCs. Time-kill studies showed reduction rates of 4log10CFU/mL for 200mg/L and 5log10CFU/mL for 400mg/L polyhexanide within 5-30min. It is concluded that polyhexanide is suitable for topical eradication of S. aureus.

The activation domain of simian immunodeficiency virus SIVmac239 Rev protein is structurally and functionally analogous to the HIV-1 Rev activation domain.

The Rev proteins of primate immunodeficiency viruses are essential transactivators for the switch from early to late phase in the viral replication cycle. By mutational analysis, a putative activation domain (AD) has been assigned to the carboxy-terminus. This leucine-rich stretch of amino acids proved to be essential for the transactivating properties of HIV-1 Rev. Some mutants in the AD transdominantly inhibit the function of wild-type Rev protein very efficiently. We identified a similar domain structure for SIVmac239 Rev by sequence comparison and in vitro mutagenesis. The leucine/isoleucine residues of the SIVmac239 Rev activation domain appeared to be of similar importance for function. The mutants of these residues in the SIV AD displayed a dominant negative phenotype on both HIV-1 and SIVmac 239 rev-responsive elements (RRE). The prokaryotically expressed wild-type and mutant proteins were analyzed for RNA-binding properties in a gel-shift assay in vitro. This assay revealed a similar binding pattern of wild-type and transdominant proteins on either RRE.

Exchange of functional domains between Rev proteins of HIV-1 and SIVmac239 results in a dominant negative phenotype.

The Rev proteins of primate immunodeficiency viruses are essential transactivators to switch from early to late phase in the viral replication cycle. Surprisingly, the Rev protein of HIV-1 is able to substitute those of HIV-2 and, as shown in here, of SIVmac239, but not vice versa. To understand the underlying mechanism of this incomplete functional reciprocity, we constructed a series of chimeric HIV-1/SIVmac239 Rev proteins and tested for transcomplementation efficacy on Rev-dependent indicator plasmids. In addition, we analyzed the prokaryotically expressed wild type and chimeric proteins for RNA-binding properties in a gel-shift assay in vitro. The functional defect of SIVmac239 on the HIV-1 Rev response element (RRE) is not due to a lack of binding or multimerization. In cotransfection experiments, SIVmac239 Rev and the chimeric proteins were tested for potential inhibitory effects on HIV-1 Rev function using the HIV-1 based indicator plasmid. Some of these proteins turned out to be transdominant inhibitors almost as potent as the HIV-1 Rev mutant M10 which is localized in the activation domain and is one of the strongest transdominant inhibitors. Surprisingly, M10 was not able to inhibit the function of either Rev protein on SIVmac239 RRE, whereas a corresponding SIVmac239 Rev mutant (SIV M10) was a transdominant inhibitor of SIVmac239 Rev function on its homologous RRE.

Disseminated fatal human cytomegalovirus disease after severe trauma.

OBJECTIVE: Disseminated human cytomegalovirus (HCMV) disease is considered to be uncommon in critically ill but otherwise not immunosuppressed patients. We describe the case of a trauma victim who developed fatal HCMV disease that initially presented as pseudomembranous colitis and resulted in sudden cardiac death. DESIGN: Case report of fatal HCMV disease in a previously healthy patient after multiple trauma. SETTING: Surgical intensive care unit (ICU). PATIENT: A 63-yr-old male patient with multiple injuries. INTERVENTIONS AND MEASUREMENTS: Under ICU treatment, symptoms of HCMV reactivation presenting as pseudomembranous colitis appeared 32 days after trauma. Detailed laboratory examinations for HCMV infection were performed, including complement fixation titer, immunoglobulin G and M, polymerase chain reaction, and virus isolation. RESULTS: The intravital detection of HCMV DNA in serum, leukocytes, and a colonic biopsy specimen indicated HCMV reactivation. Postmortem examination findings, including positive viral cultures, showed severe disseminated HCMV disease with involvement of the colon and myocardium. CONCLUSIONS: The lack of specific clinical symptoms of HCMV disease and the delay until viral culture results are available make an exact and timely diagnosis of HCMV disease difficult. Its prevalence in critically ill but otherwise not immunosuppressed patients is currently unknown and possibly underestimated. Because severe illness or trauma can cause immunodysfunction and, thus, may contribute to an increased rate of HCMV disease, detailed studies are warranted to evaluate the real risk in the ICU setting.

Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1.

The rev (art/trs) protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 K apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. The rev protein was expressed in Escherichia coli as a beta-galactosidase fusion protein with a cleavage site for proteinase factor Xa. The rev-specific fragment was isolated to immunize mice. Five stable hybridoma cell lines were obtained producing monoclonal antibodies that reacted with rev protein in Western blot and ELISA. Using the monoclonal antibodies in indirect immunofluorescence, the rev protein could be localized in the nucleus, mostly in the nucleoli, of Hela cells that were transfected with a eukaryotic rev expression plasmid.

Mouse monoclonal antibodies recognizing the activation domain of HIV-1 rev transactivator.

The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. So far, at least four necessary functional domains have been assigned to the HIV-1 rev protein: (1) one for RNA binding; (2) a second for nuclear/nucleolar localization that may be indistinguishable from the RNA binding motif; (3) two domains for multimerization; and (4) a putative activation domain (AD) that is suppressed in trans by dominant-negative mutant rev protein. We report three IgG1 kappa mouse monoclonal antibodies (mabs) that were independently raised against rev protein expressed in Escherichia coli. Epitopes are mapped by immunoprecipitation and Western blot screening with 40 different rev mutant peptides. Surprisingly, monoclonal antibodies from all three hybridomas recognized the activation domain of HIV-1 rev.

Mutational analysis of functional domains in the HIV-1 Rev trans-regulatory protein.

HIV-1 replication depends on the expression of trans-regulatory genes (tat, rev) encoded in the 3' part of the retroviral genome. HIV-1 Rev trans-activator protein allows the cytoplasmic translocation of incompletely spliced retroviral mRNA which is required for the translational switch from regulatory (Tat, Rev, Nef) to structural proteins (Gag, Pol, Env). The HIV-1 Rev regulatory protein comprises an activation domain (RAD) and a RNA binding domain (RBD). Both functional domains are not well defined and the RBD appears to overlap with the nuclear localization signal (NLS). Our mutational analysis localized the Rev protein domain important for RRE (nucleotide 7781 to 8000) binding in vitro to amino acid residues 31 to 50. Mutations in this domain always resulted in exclusion from the nucleoli. Furthermore, these mutants did not support Rev-dependent p24 Gag production in vivo. Sequences immediately upstream of this domain (RevM4, RevM19) were attenuated in their in vivo activity possibly indicating a role in Rev protein oligomerization. The observed tight correlation between subcellular localization and RNA binding in vitro indicates that this short stretch of amino acids supports two essential functions required for HIV-1 replication.

In vitro binding of human T-cell leukemia virus rex proteins to the rex-response element of viral transcripts.

Human T-cell leukemia virus (HTLV-I, HTLV-II) rex protein function is required for the cytoplasmic expression of incompletely spliced viral transcripts encoding structural proteins. The effect is mediated by a cis-acting rex-response element (RRX) which is located near the 3' end of all viral mRNAs. We show that rex polypeptides of HTLV-I and HTLV-II expressed in Escherichia coli are capable of specifically binding RRX-containing transcripts of both viruses in cell-free assays. Binding analyses with deletion variants of rex proteins revealed a domain with RNA-binding activity in the first 77 N-terminal amino acids. Removal of a basic peptide of 19 amino acids from the N terminus abrogated RNA binding, whereas a beta-galactosidase fusion protein containing this peptide bound to the RRX. These results suggest that direct binding of rex protein to the RRX is important for rex-mediated regulation of viral gene expression and that a short stretch of positively charged amino acids contributes to the specific binding of rex to its target RNA.

Human papillomavirus type 16-associated primary squamous cell carcinoma of the rectum.

Primary squamous cell carcinoma (SCC) of the colorectum is an extremely rare malignancy of unknown etiology and pathogenesis. We describe an 87-year-old man with primary SCC of the rectum. Routine histology demonstrated a squamous metaplasia-dysplasia sequence of the rectal mucosa with subsequent malignant transformation. Molecular biologic analysis using polymerase chain reaction (PCR) and in situ hybridization revealed the presence of human papillomavirus type 16 (HPV-16) DNA within metaplastic, dysplastic, and SCC lesions and in tumor-free rectal mucosa. Moreover, nested reverse-transcription PCR showed transcriptional activity of the viral E6/E7 oncogenes in tumor tissue and tumor-free rectal mucosa. By contrast, 4 typical adenocarcinomas of the rectum and their adjacent normal mucosa were found to be negative for HPV by nested PCR. In line with the well-established concept of HPV-associated anogenital carcinogenesis, our results strongly suggest an etiologic role of HPV-16 in the pathogenesis of the metaplasia-dysplasia-SCC sequence in the case described.