Mycobacterium abscessus infection in solid organ transplant recipients: report of three cases and review of the literature.

Mycobacterium abscessus is an ubiquitous organism found in the environment. This rapidly growing mycobacterium infrequently causes disease in humans; however, in immunocompromised hosts, disease can range from localized cutaneous lesions to disseminated infection. The organism is resistant to most antimycobacterial drugs and therapy can be limited by drug interactions. The exact incidence of M. abscessus infection among solid organ transplant (SOT) recipients is unknown; data are only available from previously reported cases in the literature. We describe 3 cases of M. abscessus infection in SOT recipients diagnosed within a 5-month period. One of the cases followed multi-visceral transplantation, the first such case to be reported in the literature. An epidemiological investigation did not reveal significant commonalities among the cases, and pulsed-field gel electrophoresis of genomic DNA of the case isolates confirmed their non-identity. All cases improved with antibiotic therapy, most notably with the new glycylcycline, tigecycline, along with surgical intervention in 2 of the cases. In addition, we review features and characteristics of M. abscessus infections in recipients of SOT reported in the literature from 1992 to 2008 and summarize some selected therapeutic concerns and issues related to treatment.

A case of subcutaneous Mycoleptodiscus indicus infection in a liver transplant recipient successfully treated with antifungal therapy.

Mycoleptodiscus indicus, a dematiaceous mold, occurs on the leaves of a number of different host plants and has been only recently described as a cause of human infection. Immunosuppressed individuals are at risk for developing infections with opportunistic fungal pathogens, which are a major cause of morbidity and mortality in this population. In addition, the treatment of infections caused by these fungi is frequently challenging. We report a case of M. indicus subcutaneous infection in a 51-year-old man with human immunodeficiency virus and hepatitis C co-infection, who had a liver transplant. He developed skin nodules with a sporotrichoid lymphangitic distribution. Histopathology demonstrated unusual fungal elements with angioinvasion. Mycology cultures isolated a dematiaceous mold with the characteristic curved hyaline conidia of M. indicus. Initial treatment involved a combination of amphotericin B lipid complex and voriconazole, followed by monotherapy with voriconazole. The subcutaneous lesions resolved completely after 4 months of antifungal therapy.

Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system.

A recently introduced histologic fixative (Universal Molecular Fixative [UMFIX]) has been shown to preserve macromolecules in tissue at ambient temperature. When UMFIX-exposed tissues are processed by a formalin-free, microwave-assisted rapid processing system, the resulting paraffin blocks retain good histomorphology and intact nucleic acids suitable for expression microarray analysis. Because UMFIX may be used as an alternative to formalin, the authors set out to study the effect of this new fixation and processing system on immunohistochemistry (IHC) by analyzing a range of human neoplastic and non-neoplastic specimens. Parallel slices from surgically removed specimens were fixed in formalin and UMFIX and processed in a rapid microwave-assisted tissue processor. IHC was performed following routine procedures. The staining for those antibodies that normally required antigen retrieval was carried out with and without that step. The intensity and pattern of reactions were compared in 144 tissue samples fixed by the two methods using 70 monoclonal and polyclonal antibodies. The intensity of IHC reactions for most cytoplasmic antigens was generally equal or stronger in UMFIX tissues. This was particularly true with intermediate filaments and HercepTest, where the antigen retrieval step became unnecessary. Conversely, there was a decrease in the intensity of reactions for HepPar1, bcl-2, and three nuclear antigens (Ki-67, TTF-1, and estrogen receptor). Increasing their exposure times optimized the sensitivity of the latter four antibodies. The study shows that IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens. There is an enhancement of the sensitivity of few antibodies in UMFIX-exposed tissue, rendering antigen retrieval unnecessary. This increased sensitivity may be due to the effect of eliminating formalin from fixation and processing or the microwave energy.

Antimicrobial activity of UMFix tissue fixative.

AIMS: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms. The UMFix solution was compared with 10% neutral buffered formalin. METHODS: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin. RESULTS: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses. Bacterial spores were resistant to killing by UMFix. All organisms were killed by the 10% neutral buffered formalin preparation. CONCLUSIONS: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species. The methanol content of the fixative was responsible for the killing effect of this fixative. No killing was seen when polyethylene glycol was used alone.

Kaposiform hemangioendothelioma: the first reported case in a nonhuman animal species.

Kaposiform hemangioendothelioma is a rare vascular tumor that almost exclusively occurs in children. It is a low-grade malignant neoplasm that is locally aggressive but without proven metastatic potential. Herein, we describe a case of Kaposiform hemangioendothelioma in a 10-year-old male dog. To the best of our knowledge, this is the first report of this vascular tumor occurring in a nonhuman animal species. The tumor was located on the ventromedial surface of the posterior limb. Microscopic examination of the lesion revealed a multinodular tumor with sheets of spindled endothelial cells forming vascular slits similar to Kaposi's sarcoma and peripheral tumor lobules resembling capillary hemangioma.

Infliximab monotherapy in psoriasis: a case of rapid clinical and histological response.

BACKGROUND: Psoriasis is a common chronic relapsing, inflammatory, hyperproliferative skin disorder with genetic predisposition. There is currently no experimental model for psoriasis and the pathogenesis is not fully understood. Psoriatic plaques have been shown to contain increased levels of cytokines, including tumor necrosis factor alpha (TNF-alpha). Anti-tumor necrosis factor therapy with infliximab has been shown to be highly effective in recalcitrant psoriasis. METHODS: We evaluated the efficacy and timeline of histological changes in a psoriatic plaque following infliximab infusion. A patient with severe recalcitrant plaque psoriasis was clinically and histologically assessed for improvement. RESULTS: We found rapid clinical improvement with infliximab accompanied by histopathological changes. The earliest effects were seen on neutrophils and lymphocytes whereas keratinocyte normalization was not evident at the early stages. CONCLUSION: Infliximab is not only an effective agent in the treatment of psoriasis but appears to have a very rapid onset of action.

Topical application of imiquimod 5% cream to keloids alters expression genes associated with apoptosis.

Keloids are benign mesenchymal tumours, usually present at and extending beyond the margins of sites of previous injury. It is reported that keloids display aberrant expression of apoptotic genes: TGFB1 is activated, whereas caspase 8 and 3 are not, thus indicating a block upstream in the apoptosis cascade in keloids. Interferon-alpha 2b normalizes the excessive synthesis of collagen, glycosaminoglycans and collagenase by keloidal fibroblasts, reduces recurrences following keloid excision, and enhances the expression of native p53 and apoptosis. Imiquimod, a rapid and potent inducer of interferons locally at the site of application to the skin, reduces recurrences following keloid excision and alters gene expression of markers of apoptosis in basal cell carcinoma cells. We investigated the effects with respect to the expression of apoptotic genes in keloidal tissue compared with nontreated controls of imiquimod 5% cream applied topically to keloids. Total RNA was extracted from excised keloidal tissue, cDNA probes synthesized and then hybridized to gene-specific cDNA fragments spotted on membranes. The expression levels of 96 genes involved in apoptosis, relative to cyclophilin expression, were compared in the imiquimod-treated and untreated groups. The mean ratio of expression, relative to cyclophilin of caspase 3 and DFFA were significantly enhanced. Caspase 3 was significantly downregulated and DFFA was significantly upregulated in the group of imiquimod-treated keloids (P < 0.05) compared with the untreated group of keloids. Although imiquimod is capable of altering the expression of these markers of apoptosis in keloids, their role, if any, in the therapeutic response of keloids to imiquimod requires further investigation.

Molecular characterization of a urea transporter in the gill of the gulf toadfish (Opsanus beta).

Urea excretion by the gulf toadfish (Opsanus beta) has been shown in previous studies to be a highly pulsatile facilitated transport, with excretion probably occurring at the gill. The present study reports the isolation of an 1800 base pair (kb) cDNA from toadfish gill with one open reading frame putatively encoding a 475-residue protein, the toadfish urea transporter (tUT). tUT, the first teleostean urea transporter cloned, has high homology with UTs (facilitated urea transporters) cloned from mammals, an amphibian and a shark, and most closely resembles the UT-A subfamily. When expressed in Xenopus laevis oocytes, tUT increased urea permeability (as measured by [(14)C]urea uptake) five- to sevenfold, and this permeability increase was abolished by phloretin, a common inhibitor of other UTs. Northern analysis using the 1.8 kb clone was performed to determine the tissue distribution and dynamics of tUT mRNA expression. Of six tissues examined (gill, liver, red blood cells, kidney, skin and intestine), only gill showed expression of tUT mRNA, with a predominant band at 1.8 kb and a minor band at 3.5 kb. During several points in the urea pulse cycle of toadfish (0, 4, 6, 12 and 18 h post-pulse), measured by excretion of [(14)C]urea into the water, gill mRNA samples were obtained. Expression of tUT mRNA was found to be largely invariant relative to expression of beta-actin mRNA over the pulse cycle. These results further confirm the gill localization of urea transport in the toadfish and suggest that tUT regulation (and the regulation of pulsatile urea excretion) is probably not at the level of mRNA control. The results are discussed in the context of the mechanisms of vasopressin-regulated UT-A in mammalian kidney and morphological data for the toadfish gill.

Timecourse study of UVB-induced cytokine induction in whole mouse skin.

Ever since the skin was recognized as a site of immunologic activity, a number of laboratories have studied the production of cytokines by skin cells and the effects of chemicals, allergens, contact irritants and UVB radiation on their production. Most research to date has been carried out using either purified populations of primary cells, or established cell lines. Cytokines, however, do not function in isolation but they appear in human tissues within the context of other cytokines that can, in turn, strongly influence the final biological outcome. Therefore, in vivo studies using whole skin are more physiologically relevant since all cell types are present and interactions among them are allowed to proceed. We report here the results of a detailed timecourse study using whole mouse skin, consisting of both dermis and epidermis, irradiated with either a low or high dose of UVB and analyzed using a Multi-probe RNase protection assay system. The results show that in whole skin the kinetics of cytokine induction are different than what was previously observed in tissue culture. In addition to already known skin-associated cytokines, we report here the presence and UVB induction of cytokines not previously reported.

Mapping of genetic deletions on the long arm of chromosome 22 in human pancreatic adenocarcinomas.

The molecular mechanisms of carcinogenesis in pancreatic cancer are still poorly understood, although the inactivation of tumor suppressor genes at multiple loci is suspected. We investigated the loss of heterozygosity (LOH) on chromosome 22 in pancreatic cancer by means of a PCR-based microsatellite analysis of archival paraffin-embedded histological sections in order to better define deleted region(s) and to test whether the NF-2 gene is involved. Using a panel of thirteen markers that spanned the long arm of chromosome 22, loss of heterozygosity was identified for at least one locus in 37% of investigated pancreatic adenocarcinomas. These deletions are clustered into two separate areas of the chromosome 22--one proximal to the NF-2 gene and one distal. The NF-2 gene itself is not involved. These regions are likely locations of tumor suppressor genes that may contribute to the development of pancreatic cancer.

Early cancer detection by microsatellite marker analysis.

A major goal of tumor biology has been the development of tumor markers that are useful for the diagnosis and management of cancer. A drawback associated with many of these markers is that they are not unique to tumor cells, but rather are normal or developmental antigens which are overexpressed in tumor tissue Therefore, determination of possible malignancy is based on a designated expression level rather than a clear-cut difference. Studies have shown that DNA from tumor cells has a pattern of chromosomal deletion clearly distinguishable from normal cell DNA, and more importantly, DNA from tumor cells can be detected in the serum of cancer patients. Particular chromosomal deletion patterns are associated with specific tumor types. It is hypothesized that individuals at risk for certain genetically well-characterized cancers, could be successfully screened for those cancers by a PCR-based blood test. In this way, neoplasia could be detected at earlier, more treatable stages of development.

Genetic mapping and physical cloning of UVB susceptibility region in mice.

One of the most important goals of cancer research is to identify environmental and host factors that contribute to the malignant state. Human skin cancers are among the few tumor types for which the predominant environmental carcinogen is known. Ultraviolet light, a component of sunlight, is an important cause of skin cancer in humans. In humans and mice, ultraviolet B radiation induces systematic and local immunosuppression. A consequence of that is inappropriate immune surveillance of somatic tissues for evidence of malignantly transformed cells. The impairment of contact hypersensitivity, as it develops early and correlates well with tumor frequency in various mouse strains, has been used for over 15 y as a model of immunologic events occurring in photocarcinogenesis. In mice, as well as in humans, ultraviolet B radiation induced impairment of contact hypersensitivity is not uniform in all individuals; some individuals are susceptible to the deleterious effects of ultraviolet B, whereas others are resistant to ultraviolet B. We have defined the genetic locus responsible for ultraviolet B susceptibility and resistance in mice within the Bat5 and H-2D segment of the mouse chromosome 17.

Sequence analysis and expression of a mouse homolog of human IkappaBL gene.

The family of transcriptional inhibitors, IkappaBLs, are critical to the regulation of cytokine and chemokine production. We have identified the complete cDNA sequence of the mouse IkappabL gene. The predicted 381-amino-acid sequence showed evidence of two ankyrin repeats characteristic of Ikappab family proteins and 92% identity to the IkappaBL human homolog. Although human IkappaBL has been reported to be ubiquitously expressed, here we show that mouse IkappaBL is transcribed in a more tissue-specific manner.

Interleukin 11 reduces apoptosis in UVB-irradiated mouse skin.

BACKGROUND: UVB radiation, the major cause of skin cancer, causes apoptosis in the skin, characterized by the appearance of 'sunburn cells'. Interleukin 11 (IL-11) has been shown to protect cells in the gastrointestinal system from chemotherapy and radiation-induced death. OBJECTIVE: To determine whether exogenous IL-11 has any effect on the UVB-induced apoptosis of cutaneous tissues. METHODS: Mice were injected with IL-11 or vehicle control prior to irradiation, and TUNEL assays were performed on paraffin-embedded tissue sections at various times after irradiation. RESULTS: Exogenous IL-11 protects epidermal keratinocytes, epithelial cells of the hair follicles inflammatory cells and fibroblasts in the dermis from UVB-induced apoptotic death. CONCLUSION: These findings suggest that IL-11 may play a role in the general homeostasis of the skin.

Characterization and sequencing of glutamine synthetase cDNA from liver of the ureotelic gulf toadfish (Opsanus beta).

The hepatic enzyme, glutamine synthetase (GSase) is a pivotal protein in the regulation of urea synthesis in fish. The sequence of the DNA encoding for GSase from liver of the ureotelic gulf toadfish (Opsanus beta) was analyzed through a suite of molecular techniques (including cDNA cloning, RACE PCR, and genomic PCR). An open reading frame (ORF) was identified in the cDNA sequence which codes for a protein of 394 amino acids with high identity (86%) to dogfish shark GSase. In the course of generating a suitable probe, a partial sequence was also obtained for horned shark GSase which also had high identity with the dogfish shark gene (93%). Like the dogfish shark GSase, the toadfish gene has two methionine translation initiation sites; the downstream site apparently codes for a cytoplasmic isozyme, while the upstream site adds an N-terminal peptide leader sequence of 23 amino acids to the 'cytoplasmic' protein. This leader sequence has characteristics consistent with a mitochondrial targeting peptide, including a cleavage recognition motif (Arg-X-Phe) and the apparent ability to form an amphiphathic helix. Northern analysis revealed that there is a single predominant transcript of approximately 2 kb in size. These results are consistent with the interpretation that in the gulf toadfish GSase cytoplasmic and mitochondrial isozymes are coded for by a single gene and mRNA transcript which is differentially translated at either initiation site. These results are discussed in the context of prior results for enzyme kinetic characteristics and urea synthesis/excretion physiology.

Linkage relationships and haplotype polymorphism among cichlid Mhc class II B loci.

The species flocks of cichlid fishes in the Great East African Lakes are paradigms of adaptive radiation and hence, of great interest to evolutionary biologists. Phylogenetic studies of these fishes have, however, been hampered by the lack of suitable polymorphic markers. The genes of the major histocompatibility complex hold the promise to provide, through their extensive polymorphism, a large number of such markers, but their use has been hampered by the complexity of the genetic system and the lack of definition of the individual loci. In this study we take the first substantial step to alleviate this problem. Using a combination of methods, including the typing of single sperm cells, gyno- or androgenetic individuals, and haploid embryos, as well as sequencing of class II B restriction fragments isolated from gels for Southern blots, we identify the previously characterized homology groups as distinct loci. At least 17 polymorphic class II B loci, all of which are presumably transcribed, have been found among the different species studied. Most of these loci are shared across the various cichlid species and genera. The number of loci per haplotype varies from individual to individual, ranging from 1 to 13. A total of 21 distinct haplotypes differing in the number of loci they carry has thus far been identified. All the polymorphic loci are part of the same cluster in which, however, distances between at least some of the loci (as indicated by recombination frequencies) are relatively large. Both the individual loci and the haplotypes can now be used to study phylogenetic relationships among the members of the species flocks and the mode in which speciation occurs during adaptive radiation.

Analysis of zebrafish Mhc using BAC clones.

Although major histocompatibility complex (Mhc) genes have been identified in a number of species, little is yet known about their organization in species other than human and mouse. The zebrafish, Danio rerio, is a good candidate for full elucidation of the organization of its Mhc. As a step toward achieving this goal, a commercially available zebrafish BAC library was screened with probes specific for previously identified zebrafish class I and class II genes, as well as for genes controlling the proteasome subunits LMP7 and LMP2. Restriction maps of the individual positive clones were prepared and the Mhc (LMP7) genes localized to specific fragments. The total length of genomic DNA fragments with Mhc genes was approximately 1700 kilobases (kb) (200 kb of fragments bearing class I loci and 1500 kb of fragments bearing class II loci). One of the two class I loci (Dare-UCA) is closely associated with the LMP7 locus; the second class I locus (Dare-UAA) is more than 50 kb distant from the UCA locus and has no LMP genes associated with it. None of the class II genes are linked to the class I or the LMP genes. All six of the previously identified class II B genes and one of the three class II A genes were found to be present in the BAC clones; no new Mhc loci could be identified in the library. Each of the six previously identified class II B loci was found to be borne by a separate group of BAC clones. The Dare-DAB and -DAA loci were found on the same clone, approximately 15 kb apart from each other. An expansion of DCB and DDB loci was detected: the zebrafish genome may contain at least five closely related DCB and two closely related DDB loci which are presumably the products of relatively recent tandem duplication. These results are consistent with linkage studies and indicate that in the zebrafish, the class I and class II loci are on different chromosomes, and the class II loci are in three different regions, at least two of which are on different chromosomes.

Linkage of TATA-binding protein and proteasome subunit C5 genes in mice and humans reveals synteny conserved between mammals and invertebrates.

The TATA-binding protein (TBP) is a factor required for the transcription of all classes of eukaryotic genes. Here, we demonstrate that in the mouse the TBP-encoding gene (Tbp) resides next to the proteasomal subunit C5-encoding gene (Psmb1). The genes are located on mouse chromosome 17 in the t complex within the Hybrid sterility 1 (Hst1) region. We demonstrate that the homologous human genes (TBP AND PSMB1) are tightly linked on the long arm of chromosome 6, in a region syntenic with the proximal part of mouse chromosome 17. The mouse Tbp and Psmb1 and the human TBP and PSMB1 genes are transcribed in the opposite orientation. The TATA-binding protein and proteasomal subunit C5 genes are also linked on chromosome III of Caenorhabditis elegans, and together they are linked to other genes whose homologs map to human chromosome 6 and mouse chromosome 17. In the Drosophila genome, the housekeeping TATA-binding protein gene maps close to two other genes with homologs in the mammalian major histocompatibility complex. There thus exists conserved synteny of unrelated genes between mammals and invertebrates.

Isolation of candidate hybrid sterility 1 genes by cDNA selection in a 1.1 megabase pair region on mouse chromosome 17.

The Hybrid sterility 1 (Hst1) gene causes male infertility in crosses between certain inbred strains of the laboratory and wild mouse, Mus musculus. To identify the causative gene, we have searched YAC clones encompassing the Hst1 region for testis-expressed sequences, using the cDNA selection method. We isolated 12 non-overlapping cDNA clones, sequenced them, and placed them on a physical map based on the analysis of YAC clones and total genomic DNA. The cDNA clones map to ten loci. Three cDNA sequences correspond to the proteasome subunit C5 (locus Psmb1), ornithine decarboxylase (Odc-rs15), and penta-zinc finger (Zfp91-rs1) transcripts. Three of the ten testis-expressed loci described in this report (D17Ph4e, Psmb1, and Zfp91-rs1) co-segregate with all Hst1 recombinants and, together with the Tbp gene, are therefore potential candidates for the Hst1 gene. The presented physical and genetic mapping data indicate there are no gross rearrangements distinguishing the Hst1(f) and Hst1(s) alleles.