High abundance of human herpesvirus 8 in wastewater from a large urban area.

AIMS: This study assesses the diversity and abundance of Human Herpesviruses (HHVs) in the influent of an urban wastewater treatment plant using shotgun sequencing, metagenomic analysis and qPCR. METHODS AND RESULTS: Influent wastewater samples were collected from the three interceptors that serve the City of Detroit and Wayne, Macomb and Oakland counties between November 2017 to February 2018. The samples were subjected to a series of processes to concentrate viruses which were further sequenced and amplified using qPCR. All nine types of HHV were detected in wastewater. Human Herpesvirus 8 (HHV-8), known as Kaposi's sarcoma herpesvirus, which is only prevalent in 5-10% of USA population, was found to be the most abundant followed by HHV-3 or Varicella-zoster virus. CONCLUSIONS: The high abundance of HHV-8 in the Detroit metropolitan area may be attributed to the HIV-AIDS outbreak that was ongoing in Detroit during the sampling period. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach described in this paper can be used to establish a baseline of viruses secreted by the community as a whole. Sudden changes in the baseline would identify changes in community health and immunity.

Pilot study to assess the effects of early flea exposure on the development of flea hypersensitivity in cats.

This pilot study was to determine if early oral flea exposure reduces the incidence of flea allergy dermatitis (FAD) in cats. Eighteen kittens, assigned to three groups, received no flea exposure, oral flea exposure or flea infestation for 12 weeks. Then all the kittens were exposed continually to fleas for 31 weeks. Sensitization was monitored using intradermal testing (IDT), in vitro measurement of anti-flea saliva immunoglobulin E (IgE) and development of FAD. There was no statistically significant difference between groups in IDT reactions, in vitro data or clinical scores. The development of FAD was not associated with the presence of anti-flea saliva IgE. However, the development of a delayed reaction to flea bite was associated with symptoms after flea exposure. Although not statistically significant, the FAD scores in the oral group were lower than in the controls. Further studies are required to determine the role of oral flea exposure in the development of FAD in cats.

Are keloids really "gli-loids"?: High-level expression of gli-1 oncogene in keloids.

BACKGROUND: Keloids are a common lesion arising from sites of previous trauma and are a considerable source of morbidity because of continued growth of lesions, pruritus, and physical appearance. They consist of mesenchymal cells embedded in a stroma of disordered collagen matrix. Clinically, keloids are distinguished from scars in that keloids demonstrate continued growth over the borders of the original injury. Keloids appear with increased frequency in patients of African and Asian descent. Currently, no entirely satisfactory method of treatment exists for these lesions. Recently, a patient who was enrolled in a clinical trial of topical tacrolimus for atopic dermatitis applied this drug to a keloid and noted clearing. OBJECTIVE: Based on this clinical observation and the observation that rapamycin, a chemically similar compound to tacrolimus, is known to inhibit signaling from the gli-1 oncogene, we examined keloids and scars for expression of Gli-1 protein. METHODS: Skin sections from keloids and scars were examined by immunohistochemical staining for gli-1. To further confirm the presence of gli-1 expression in keloids, reverse transcriptase-polymerase chain reaction was carried out. RESULTS: Expression of gli-1 was strongly elevated in keloids compared with scars. CONCLUSION: These results provide a rationale for the treatment of keloids with topical rapamycin analogs, including tacrolimus. Clinical trials of topical tacrolimus are warranted.

Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs.

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.

Canine interleukin-5: molecular characterization of the gene and expression of biologically active recombinant protein.

Interleukin-5 (IL-5), which is produced primarily by type 2 T helper lymphocytes (Th2), is an eosinophil differentiation and activation factor. Increased numbers of eosinophils in peripherial blood or tissues (eosinophilia) are observed in asthmatic human patients, in animals with helminth infections, and in dogs with allergic diseases. Antagonism of IL-5 activity is being explored as a potential treatment of a number of disease conditions associated with eosinophils in animal models. In order to study the expression and function of this cytokine in the dog, we have isolated and characterized the canine IL-5 gene. The canine IL-5 polypeptide deduced from the cDNA is composed of 134 amino acids that share varying degrees of homology with IL-5 isolated from several mammals. The genomic structure of the canine IL-5 gene consists of four exons and three introns in the coding region, similar to that of the previously characterized human and mouse IL-5 genes. Recombinant canine IL-5 protein, expressed in Pichia pastoris, is biologically active in a cell proliferation assay. Canine IL-5 gene sequences and the biologically active protein described in this study will be useful reagents for future studies of this cytokine in physiologic processes and in pathologic conditions of the dog.

Cloning and characterization of cDNAs encoding four different canine immunoglobulin gamma chains.

cDNAs encoding four different canine immunoglobulin G (caIgG) gamma chains were identified in this study. One of these IgG gamma chain cDNAs, (caIgG-A), represents 92.5% of the IgG gamma chain cDNAs in a dog spleen cell cDNA library; a second partial IgG gamma chain cDNA (caIgG-B) was also identified in the library. The other two IgG gamma chain cDNAs (caIgG-C and caIgG-D) were RT-PCR amplified from canine lymphoma samples. Comparison of the four different canine IgG gamma chain cDNAs showed homologies from 83.6 to 89.2% and from 73.1 to 81.8% at nucleotide and amino acid sequence levels, respectively. Despite the high similarity in CH1, CH2 and CH3 domains among the different caIgG gamma chains, the hinge regions were distinct, sharing only 19.0-35.2% homology at the amino acid level. No multiple duplication of the hinge region, as reported for human IgG1 and IgG3, was detected in any of the canine IgG gamma chains. The numbers of cysteines in the putative hinge regions were found to be 3, 2, 7 and 3 for the four canine IgG heavy gamma chains (A, B, C and D), respectively. Specific primers were designed based on caIgG gamma chain hinge region DNA sequences and were used in RT-PCR for measuring different caIgG gamma chain mRNA levels in canine PBMC samples.

Endotoxin inducible transcription is repressed in endotoxin tolerant cells.

Stimulation of the human promonocytic cell line, THP-1, with endotoxin results in a rapid and transient increase in interleukin 1beta expression. Endotoxin pretreatment of THP-1 cells results in tolerance, characterized by decreased levels of endotoxin-induced interleukin 1beta expression due to decreased transcription of the interleukin 1beta gene. We hypothesized that tolerant cells could not activate transcription factors necessary to express the interleukin 1beta gene. This hypothesis was tested in tolerant THP-1 cells by using stable and transiently transfected reporter genes containing the interleukin 1beta promoter. We found decreased endotoxin-induced transcription of all reporter genes tested; however, individual transcription factors, such as NFkappaB, retain normal, CD14-dependent, nuclear translocation and DNA binding. Tolerance is specific for endotoxin, because phorbol ester is still able to activate transcription of the endogenous interleukin 1beta gene and transfected reporter genes. A constitutively active reporter gene that is not inducible by endotoxin is unaffected. We further show that nuclear extracts of tolerant cells show transcription inhibitor activity that is specific for promoter sequences of the interleukin 1beta gene. These results support a mechanism of endotoxin tolerance that is independent of transcription factor DNA binding and appears to be associated with the inability of DNA-bound transcription factors to activate transcription, perhaps through the activity of a repressor.

Molecular mechanisms responsible for endotoxin tolerance.

Bacterial lipopolysaccharide endotoxin (LPS) is a potent activator of a number of inflammatory genes, including interleukin-1 (IL-1). IL-1 and other cytokines such as tumor necrosis factor alpha (TNF alpha) are essential mediators in inducing severe sepsis syndromes (SS). Major cellular targets of LPS are blood or tissue leukocytes, such as macrophages and neutrophils. These cells can respond and adapt to LPS, the latter phenomenon is known as LPS tolerance. In animals, LPS tolerance is a highly effective mechanism of protection against the lethal syndrome of severe sepsis. Two models are used to investigate the molecular basis of LPS tolerance. The first model employs blood leukocytes isolated from patients with SS. The second model employs the promonocytic cell line, THP-1 in vitro. In the SS model, LPS tolerance of involves repression at the level of IL-1 beta mRNA. Suppression of IL-1 beta mRNA is under the control of a labile repressor protein. In contrast to suppression of IL-1 beta, mRNA is under the control of a labile repressor protein. In contrast to suppression of IL-1 beta, there is increased expression of the Type 2 IL-1 receptor mRNA and protein in leukocytes from patients with SS. The THP-1 model of LPS tolerance also involves repression of LPS induction of IL-1 beta gene expression. The repression of THP-1 cell IL-1 beta expression is at the level of transcription, and like the SS model is under the control of a labile protein. LPS tolerance in both models is stimulus-specific. We further find that transcription factors such as NF kappa B and AP-1 may participate in regulating LPS tolerance.

Sporotrichoid cutaneous leishmaniasis in a traveler.

A 19-year-old construction worker from Virginia who had traveled in Bolivia had sporotrichoid lesions on the left arm. Only after unsuccessful therapy for sporotrichosis was a diagnosis of cutaneous leishmaniasis considered. Biopsies revealed necrotizing granulomatous changes, and culture of the biopsy specimens grew Leishmania (Viannia) braziliensis. The sporotrichoid pattern seen in this patient is a rare but recognized presentation of cutaneous leishmaniasis, more commonly seen in American cutaneous leishmaniasis than in Old World cutaneous leishmaniasis. This case illustrates the necessity of careful and early consideration of tropical infections in the differential diagnosis of disease in a traveler.

Protein-tyrosine kinase activation is required for lipopolysaccharide induction of interleukin 1beta and NFkappaB activation, but not NFkappaB nuclear translocation.

In human monocytes, interleukin 1beta protein production and steady state mRNA levels are increased in response to lipopolysaccharide, predominantly as a result of increased transcription of the interleukin 1beta gene. Expression of interleukin 1beta and other cytokines, such as interleukin 6 and tumor necrosis factor alpha, has been shown to be dependent on the activation of the transcription factor, NFkappaB. Since recent studies have shown that lipopolysaccharide-induced tyrosine kinase activation is not required for NFkappaB nuclear translocation, we sought to determine whether NFkappaB translocated in the absence of tyrosine kinase activity was active in stimulating transcription. We have found that, in the human pro-monocytic cell line, THP-1, the lipopolysaccharide-induced expression of interleukin 1beta is dependent on tyrosine kinase activation. Tyrosine kinases are not required for lipopolysaccharide-mediated nuclear translocation of NFkappaB. However, in the absence of tyrosine kinase activity, the ability of NFkappaB to stimulate transcription is impaired. This inhibition of transcription is specific for NFkappaB; in the absence of tyrosine kinase activity, AP-1-dependent transcription is enhanced. These results suggest that, while lipopolysaccharide-induced expression of inflammatory mediators requires tyrosine kinase activity, tyrosine kinase activity is not obligatory for lipopolysaccharide signal transduction.

Antitumor benzothiazoles. 3. Synthesis of 2-(4-aminophenyl)benzothiazoles and evaluation of their activities against breast cancer cell lines in vitro and in vivo.

A new series of 2-(4-aminophenyl)benzothiazoles substituted in the phenyl ring and benzothiazole moiety has been synthesized by simple, high-yielding routes. The parent molecule 5a shows potent inhibitory activity in vitro in the nanomolar range against a panel of human breast cancer cell lines, but is inactive (IC50 > 30 microM) against other cell types: activity against the sensitive breast lines MCF-7 and MDA 468 is characterized by a biphasic dose-response relationship. Structure-activity relationships derived using these cell types has revealed that activity follows the heterocyclic sequence benzothiazole > benzoxazole >> benzimidazole and that 2-(4-aminophenyl)benzothiazoles bearing a 3'-methyl- 9a, 3'-bromo- 9c, 3'-iodo- 9f, and 3'-chloro-substituent 9i are especially potent and their activity extends to ovarian, lung, and renal cell lines. Four compounds have been evaluated in vivo against human mammary carcinoma models in nude mice. Compound 9a showed the most potent growth inhibition against the ER+ (MCF-7 and BO) and ER- (MT-1 and MT-3) tumors. Our efforts to identify a pharmacological mechanism of action for these intriguing compounds have not, as yet, been successful.

Elevated levels of shed type II IL-1 receptor in sepsis. Potential role for type II receptor in regulation of IL-1 responses.

Two types of cellular IL-1Rs have been characterized and cloned from both human and murine sources. The type II IL-1R has a very short cytoplasmic domain and does not seem to participate in IL-1 signaling. We demonstrate that type II IL-1Rs are released from the surface of neutrophils in response to treatment with TNF or endotoxin. In addition, serum from patients with sepsis syndrome contains elevated levels of soluble type II IL-1Rs. Neutrophils isolated from patients with sepsis have greatly enhanced expression of type II IL-1R mRNA and cell surface receptors and are therefore a likely source for the shed receptors in serum. Of the three forms of IL-1, soluble type II IL-1R binds IL-1 beta with highest affinity and also selectively inhibits IL-1 beta activity. We propose that increased cell surface expression and rapid release of preformed type II IL-1R from neutrophils, as a soluble IL-1 beta binding protein, represents a mechanism that has evolved for regulating IL-1 activity in sepsis.

A labile transcriptional repressor modulates endotoxin tolerance.

Tolerance to bacterial lipopolysaccharide (LPS, endotoxin) is an adaptive cellular process whereby exposure to endotoxin induces a subsequent hyporesponsive state characterized by decreased levels of LPS-induced cytokine mRNA and protein. We demonstrate, in a human promonocytic cell line, THP-1, that endotoxin tolerance is manifested by decreased LPS-induced interleukin 1 beta (IL-1 beta) transcription. Inhibition of protein synthesis reverses the tolerant phenotype by inducing transcription of IL-1 beta in the absence of a second stimulus. These results indicate that a labile protein contributes to the endotoxin-tolerant phenotype, and that this factor acts in a dominant repressive manner to inhibit the activity of existing transcription factors. We provide further data that cellular expression of I kappa B-alpha correlates with downregulated IL-1 beta gene expression during endotoxin tolerance, implicating I kappa B-alpha as a potential candidate for the labile repressor identified herein.

Structural studies on bioactive compounds. 23. Synthesis of polyhydroxylated 2-phenylbenzothiazoles and a comparison of their cytotoxicities and pharmacological properties with genistein and quercetin.

A series of polyhydroxylated 2-phenylbenzothiazoles 3 has been prepared by demethylation of the precursor methoxylated 2-phenylbenzothiazoles 9. The key step in the construction of the benzothiazole nucleus involves a Jacobson cyclization of methoxylated thiobenzanilides 8. The target compounds inhibit WiDr human colon tumor cells and MCF-7 human mammary tumor cells in vitro with IC50 values in the low micromolar range, but the activity against MCF-7 cells is not related to estrogen receptor-binding affinity. None of the compounds showed selective cytotoxicity against Abelson virus-transformed ANN-1 cells encoded with the pp120gag-abl tyrosine kinase compared with the parental 3T3 line. Compounds were only marginally inhibitory to the EGF receptor-associated protein tyrosine kinase from a membrane preparation of A431 cells. The most active compound was 4,6-dihydroxy-2-(4-hydroxyphenyl)benzothiazole (3b) which has the same overall hydroxyl substitution pattern as genistein (1a). The compounds were weakly cytotoxic for an EGF receptor, overexpressing cell line HN5, but when tested for differential toxicity against the EGF receptor tyrosine kinase or the PDGF receptor tyrosine kinase in a standard mitogenesis assay utilizing human fibroblasts, no discrimination was observed. In this assay, the compounds inhibited DNA synthesis when added to cells during S phase. This suggests that inhibition could not be interpreted in terms of tyrosine kinase inactivation but more likely as a relatively broad specificity for the ATP-binding domain of other kinases such as thymidine kinase.

A membrane vesicle/ribosome preparation from Serratia marcescens elicits peritoneal exudate cells expressing both tumoricidal and bactericidal activity.

A biological response modifier called ImuVert, derived from the bacterium Serratia marcescens, produced long-lasting elevation of peritoneal exudate cell (PEC) numbers after intraperitoneal injection into mice. These cells had enhanced ability to phagocytose both latex beads and opsonized Listeria monocytogenes. PEC harvested 2-14 days after a single injection of ImuVert killed L. monocytogenes, and ImuVert protected mice from infection by L. monocytogenes, measured both by LD50 and bacterial growth in vivo. Cells harvested 7 and 14 days after ImuVert injection also were tumoricidal, measured as killing of P815 mastocytoma cells, and ImuVert induced macrophages to express tumoricidal properties in vitro. These data suggest that ImuVert has a unique ability to induce a chronic inflammatory response, as other agents do not induce such a long-lasting influx of bactericidal inflammatory cells that also show tumoricidal activity. The consequences of this response appear to include protection from infection by certain bacteria.

Adenovirus E1A makes two distinct contacts with the retinoblastoma protein.

Two regions near the amino terminus of the adenovirus E1A protein, which were first identified by sequence conservation among various adenovirus serotypes, have been shown by genetic studies to be essential for E1A-mediated transformation. These same regions are also required for interaction with a number of cellular proteins, including the retinoblastoma protein (pRB). Using synthetic peptides corresponding to portions of these conserved regions, we show that each region can bind independently to pRB. These interactions were observed in both competition and binding assays. In both types of assay, region 2 peptides (E1A amino acids 115 to 132) bound pRB with higher affinity than did region 1 peptides (E1A amino acids 37 to 54), while a peptide combining region 1 and 2 sequences consistently provided the highest-affinity interaction. Cross-blocking experiments using region 1 peptides and region 2 peptides suggested that these two regions of E1A make distinct contacts with pRB. These data support the notion that the pRB-binding domain of E1A contains at least two functional elements.

The myelopoietic effects of a Serratia marcescens-derived biologic response modifier in a mouse model of thermal injury.

The proliferative defects observed in phagocytic stem cells after major thermal injuries may be caused by an inadequate production of colony-stimulating factors (CSFs), a family of hemopoietic cytokines necessary for the production and function of granulocytes and monocytes. In this study a biologic response modifier (S-BRM) consisting of sized vesicles derived from the cell membrane and ribosomes of Serratia marcescens was investigated in a mouse model of thermal injury to determine its ability to augment postburn myelopoiesis. Treatment of burned mice with S-BRM was well tolerated and was associated with statistically significant increases in absolute numbers of circulating granulocytes and monocytes compared with burned mice receiving saline solution. In addition, the size of the splenic myeloid stem cell compartment, as measured by granulocyte-macrophage stem cell colony formation in soft agar, was markedly expanded. Finally, plasma levels of CSF were increased significantly in burned mice receiving S-BRM but were not elevated in burned littermates treated with saline solution. These data suggest that production of CSF is suboptimal after thermal injury and S-BRM is capable of up-regulating postburn myelopoiesis by causing the release of CSF into the systemic circulation.