Microbiomes as sources of emergent host phenotypes.

Microbial communities associated with animals exert powerful influences on host physiology, regulating metabolism and immune function, as well as complex host behaviors. The importance of host-microbiome interactions for maintaining homeostasis and promoting health raises evolutionarily complicated questions about how animals and their microbiomes have coevolved, and how these relationships affect the ways that animals interact with their environment. Here, we review the literature on the contributions of host factors to microbial community structure and corresponding influences of microbiomes on emergent host phenotypes. We focus in particular on animal behaviors as a basis for understanding potential roles for the microbiome in shaping host neurobiology.

Viral dynamics of early HIV infection in neonatal macaques after oral exposure to HIV-2287: an animal model with implications for maternal-neonatal HIV transmission.

A model of vertical HIV transmission was developed using oral HIV-2(287) exposure of newborn Macaca nemestrina. The minimal Animal Infectious Dose for this oral route was found to be 10-fold higher than that for atraumatic viral transmission across other mucosal membranes (vaginal/rectal) of juvenile macaques. However, once infection was established, viral replication was rapid and plasma viremia could be detected by reverse-transcriptase polymerase chain reaction and viral co-culture within 1 week following exposure. No animal was resistant to infection and all macaques initially exposed to a subinfectious viral inoculum were subsequently infected by re-exposure of mucosal membranes. Higher viral load during primary infection correlated with a more rapid CD4 depletion; however, all HIV-2(287)-infected animals developed CD4 depletion during the observation period. This animal model can now be used to study early viral replication in the presence and absence of anti-retroviral agents to help identify conditions to reduce vertical HIV transmission in human newborns.

Interleukin-1 receptor antagonist as an adjunct in the treatment of Haemophilus influenzae otitis media in the chinchilla.

OBJECTIVES: This purpose of this study was to investigate the effect of blockade of the inflammatory cytokine pathway on experimentally induced otitis media in the chinchilla model. STUDY DESIGN: Pilot, randomized placebo-controlled trial. METHODS: Ampicillin-sensitive Haemophilus influenzae otitis media was induced in 45 adult chinchillas. The animals were randomly assigned to the following treatment groups: 1) transbullar injections (TBI) of interleukin-1 receptor antagonist (IL-1ra) and intramuscular ampicillin, 2) TBI of saline and intramuscular ampicillin, 3) TBI of IL-1ra and intramuscular sa-1 line or 4) TBI of saline and intramuscular saline. Blinded investigators measured resolution of otitis media by otomicroscopy, tympanogram, and culture results. RESULTS: Comparisons were made between the treatment groups to assess the ability of IL-1ra to assist with resolution of otitis media using exact two-group binomial tests with the StatXact statistical program. The group with TBI of IL-1ra and intramuscular ampicillin as a treatment demonstrated trends suggesting more rapid resolution of positive cultures and more rapid and complete return to normal results on tympanograms and otomicroscopic findings compared with the group treated with TBI of saline and intramuscular ampicillin. These trends did not achieve statistical significance with the relatively small sample sizes used in this pilot study. CONCLUSIONS: This investigation provides further evidence that the inflammatory cytokine cascade plays a significant role in the pathophysiology of otitis media and that modulation of this inflammatory pathway may provide novel and efficacious treatments for otitis media Further studies with larger groups of animals are warranted to determine whether the trends identified in this pilot study are reproducible and achieve statistical significance.

Antigen-specific cytokine responses in vaccinated Macaca nemestrina.

We describe a new surrogate assay for CD8 + T lymphocyte activity that has the capability of discriminating between cytotoxic T lymphocyte (CTL) activity and cytokine-mediated suppressive activity. We applied this approach to two groups of Macaca nemestrina vaccinated with a minimally pathogenic strain of human immunodeficiency virus type 2 [HIV-2 (HIV-2(KR))] as a model of an attenuated virus vaccine. Group 1 was then inoculated with a non-infectious stock of a pathogenic strain, HIV-2287. Both groups 1 and 2 were subsequently challenged with an infectious stock of HIV-2287. Five out of six group 1 animals were protected against CD4 decline, whereas three out of six animals in group 2 were protected. Analysis of CTL responses demonstrated strong activity against HIV-2(KR)-Gag in group 1. It was determined that strong CTL responses correlate with antigen-specific T-helper (Th) type 1 responses. This antigen-specific cytokine assay has the potential to better elucidate the functional mechanisms of CD8 + T-cell-mediated protection than traditional methods to date.

Immunization against SIVmne in macaques using multigenic DNA vaccines.

All structural and regulatory genes of SIVmne were cloned into mammalian expression vectors to optimize expression in vitro and immunogenicity in mice. Macaca fascicularis were immunized four times with plasmid DNA (n = 4), or two DNA priming inoculations followed by two boosts of recombinant gp160 plus Gag-Pol particles (n = 4). Following intrarectal challenge with SIVmne, all macaques became infected. Three monkeys immunized with DNA alone maintained low plasma virus loads by 1 year post-challenge; the fourth exhibited high virus loads and significant CD4+ cell decline. Two of the DNA plus boost and three control macaques had high virus loads and associated CD4+ cell decline. Both vaccine protocols elicited antibodies and comparable helper T-cell proliferative responses to gp160. Cytokine mRNA levels in activated peripheral blood mononuclear cells (PBMC) taken at time of challenge suggested a dominant T helper (Th) 1 state in three DNA-immunized and one protein-boosted macaque, which correlated with low virus loads and high CD4+ cell counts post-challenge.

Use of mediastinoscopy for foreign body removal.

Foreign body removal from the aerodigestive tract can be a challenging endeavor despite improvements in technology. Rigid bronchoscopy has been demonstrated to be a safe and effective means of airway foreign body removal with appropriate training and expertise. However, potential complications exist and include extraluminal impaction of a penetrating foreign body during removal. This report details such a complication and the first known use of mediastinoscopy to remove the impacted foreign body to avoid the need for thoracotomy.

Protection from pathogenic SIV challenge using multigenic DNA vaccines.

To assess DNA immunization as a strategy for protecting against HIV infection in humans, we utilized SIVmne infection of Macaca fascicularis as a vaccine challenge model with moderate pathogenic potential. We compared the efficacy of DNA immunization alone and in combination with subunit protein boosts. All of the structural and regulatory genes of SIVmne clone 8 were cloned into mammalian expression vectors under the control of the CMV IE-1 promoter. Eight M. fascicularis were immunized twice with 3 mg of plasmid DNA divided between two sites; intramuscular and intradermal. Four primed macaques received a further two DNA immunizations at weeks 16-36, while the second group of four were boosted with 250 microg recombinant gp160 plus 250 microg recombinant Gag-Pol particles formulated in MF-59 adjuvant. Half of the controls received four immunizations of vector DNA; half received two vector DNA and two adjuvant immunizations. As expected, humoral immune responses were stronger in the macaques receiving subunit boosts, but responses were sustained in both groups. Significant neutralizing antibody titers to SIVmne were detected in one of the subunit-boosted animals and in none of the DNA-only animals prior to challenge. T-cell proliferative responses to gp160 and to Gag were detected in all immunized animals after three immunizations, and these responses increased after four immunizations. Cytokine profiles in PHA-stimulated PBMC taken on the day of challenge showed trends toward Thl responses in 2/4 macaques in the DNA vaccinated group and in 1/4 of the DNA plus subunit vaccinated macaques; Th2 responses in 3/4 DNA plus subunit-immunized macaques; and Th0 responses in 4/4 controls. In bulk CTL culture, SIV specific lysis was low or undetectable, even after four immunizations. However, stable SIV Gag-Pol- and env-specific T-cell clones (CD3+ CD8+) were isolated after only two DNA immunizations, and Gag-Pol- and Nef-specific CTL lines were isolated on the day of challenge. All animals were challenged at week 38 with SIVmne uncloned stock by the intrarectal route. Based on antibody anamnestic responses (western, ELISA, and neutralizing antibodies) and virus detection methods (co-culture of PBMC and LNMC, nested set PCR- of DNA from PBMC and LNMC, and plasma QC-PCR), there were major differences between the groups in the challenge outcome. Surprisingly, sustained low virus loads were observed only in the DNA group, suggesting that four immunizations with DNA only elicited more effective immune responses than two DNA primes combined with two protein boosts. Multigenic DNA vaccines such as these, bearing all structural and regulatory genes, show significant promise and may be a safe alternative to live-attenuated vaccines.

Multicenter trial to evaluate the safety and potential efficacy of pooled human fibrin sealant for the treatment of burn wounds.

OBJECTIVE: The primary purpose of this multicenter study was to evaluate the safety and potential efficacy of a solvent/detergent-treated commercial fibrin sealant (human) for topical hemostasis in skin grafting. METHODS: The study involved a prospective evaluation of changes in viral titers in patients with burns less than 15% after treatment with fibrin sealant (human). Each patient served as his/her own control for an unblinded, randomized comparison of donor site hemostasis and healing. Preoperative serum was obtained to screen for viral titers. At autografting, the recipient site and one of two randomly chosen donor sites were treated with fibrin sealant (human). The use of other hemostatic agents, including epinephrine was prohibited. Each donor site was covered with gauze to collect blood for estimation of the relative amount of bleeding. The healing of the graft and donor sites was observed. Viral titers and wounds were checked monthly for 6 months, and at 9 and 12 months postoperatively. RESULTS: Viral titers for human immunodeficiency virus; hepatitis A, B, and C; Epstein-Barr virus; and cytomegalovirus were obtained before and after treatment. Of 47 patients, 34 completed the full year of observation. After treatment, there were no seroconversions to any of the aforementioned viruses. Bleeding at the recipient site appeared well controlled with fibrin sealant (human). Although investigators felt that fibrin sealant (human) improved donor site hemostasis, differences in hemoglobin measurements of blood-soaked dressings failed to reach significance. No differences were noted with regard to acceleration of donor site healing, graft take, or scar maturation at the two groups of donor sites. Anecdotally, the maturation of the recipient site appeared to be accelerated. CONCLUSION: Fibrin sealant (human) is safe for use during excision and grafting, and its topical hemostatic potential needs to be examined in patients with larger burns. Its role in scar maturation also needs to be investigated.

Titanium tetrachloride: an unusual agent with the potential to create severe burns.

Titanium tetrachloride (TiCl4), an intermediate compound in the production of white pigment, can cause severe burns. Two cases are reported in which TiCl4 created 18% to 20% total body surface area burns. These full-thickness injuries were the combined consequence of hydrochloric acid and the heat that was generated in areas where this otherwise stable compound was mixed with perspiration. TiCl4 combined with water is extremely dangerous, and its immediate treatment--towel drying before irrigation--makes it unique among chemicals. Our experience suggests that in most cases grafting will be required. These chemical burns were self-limited and had no notable systemic sequelae. Wound biopsy specimens taken on postburn days 3 and 6 were subjected to immunostaining that showed that TiCl4 did not retard wound healing. Exposure time to TiCl4 vapor will determine the pulmonary and ophthalmologic involvement in each case. Clinical awareness of the propensity of TiCl4 to react with water--even when that water is in the form of perspiration--is vital because prompt management can limit the extent of injury.

Progressive burn injury documented with vimentin immunostaining.

Precise determination of burn depth during the immediate postburn period remains an unresolved clinical problem. In an attempt to provide a new clinical option to aid in diagnosis of burn depth, an immunohistochemical marker (antivimentin) was used to examine excisional tissues or serial punch biopsies, or both, in partial-thickness human burn injuries. To test the hypothesis that burn injury continues to progress beyond the first 24 hours, burn depth was assessed by quantitative morphometric analysis in both a partial-thickness porcine burn model and in sequential samples from human patients. Vimentin immunostaining of ubiquitous mesenchymal populations resulted in a precise demarcation between burn eschar and the viable underlying dermis at 1 to 5 days after burn trauma. Porcine wounds showed continuous and significant progression in burn depth during days 1 through 3, but wounds were no deeper on the fourth postburn day. Similarly, 13 of 14 patients showed significant progression in burn depth between 1 to 5 days after burn injury. In conclusion, immunohistochemical staining with an antisera targeted toward a widely dispersed cell population in the dermis can be utilized as an effective tool to confirm the depth of tissue injury during the acute postburn period. Data from our randomly selected patients with partial-thickness burn suggest that burn wounds continue to demarcate for several days.

Reaction of NO with the reduced R2 protein of ribonucleotide reductase from Escherichia coli.

The active R2 protein of ribonucleotide reductase from Escherichia coli contains a catalytically essential tyrosine radical at position 122 (Tyr122.) that is formed during the reaction of dioxygen with the nearby diiron(II) center. To gain insight into the mode of dioxygen binding, the reaction of the O2 analog NO with the diiron(II) centers of R2red has been investigated by spectroscopic methods. R2red reacts with NO to form an adduct with visible absorption features at 450 and 620 nm and Mössbauer parameters (delta = 0.75 mm/s, delta EQ = -2.13 and -1.73 mm/s) typical of those observed for S = 3/2 [FeNO]7 complexes of other non-heme iron proteins. However, unlike other non-heme [FeNO]7 complexes, this adduct is EPR silent. Our Mössbauer studies show that each iron site of R2red binds one NO to form local S = 3/2 [FeNO]7 centers which then couple antiferromagnetically (J approximately 5 cm-1, H = JS1.S2) to afford an [FeNO]2 center (77% of total iron). This [FeNO]2 center decomposes with a first-order rate constant of 0.013 min-1 to form R2met, accompanied by the release of N2O. These observations suggest that both iron(II) ions of the two diiron(II) centers of R2red have available sites for NO binding, in agreement with the crystallographic results on R2red, and that the bound NO molecules are sufficiently close to each other to permit N-N bond formation to produce N2O. These observations support the proposal that dioxygen binding may also involve both metal ions of the diiron(II) center to form a (mu-1,1-, or mu-1,2-peroxo)-diiron(III) center. This observed reactivity of R2red with NO may contribute to the in vivo inhibition of ribonucleotide reductase by NO.

Is aggressive surgical management justified in the treatment of Merkel cell carcinoma?

Since its original description in 1972, we have seen and personally treated a group of 15 patients with Merkel cell carcinoma at the Vanderbilt Medical Center and the Nashville VA Hospital. We will review the demographics, management, and clinical course of this extremely lethal but initially benign appearing cutaneous malignancy. The majority of lesions occur on the head and neck, followed by the extremities and trunk. Location of the primary tumor has no effect on outcome. Despite a high mortality in our series (10 of 15), early recognition and aggressive surgical therapy may be the only way to prolong survival. No other adjuvant therapy has proved effective.

Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair.

Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair.

Burns in alcohol and drug users result in longer treatment times with more complications.

Brezel, Kassenbrock, and Stein (J Burn Care Rehabil 1988;9:169-71), surveyed a group of 180 patients who were substance abusers or who were neurologically or mentally impaired and found that their inpatient care was more costly, more complicated, and more lengthy. In our institution we undertook a similar study to determine whether our findings would be similar. During a 12-month period, the charts of all patients over the age of 18 who were admitted for treatment of acute burns were reviewed. One hundred and thirty-one charts were available for study. Review of the charts revealed that 19 (14.5%) met our criterion of being impaired by drugs or alcohol, and the remaining 112 patients served as control subjects. The total body surface area burn averaged 25.8% in the impaired group and 21.3% in the control group. The amount of third-degree burns averaged 11% in the impaired group and 11% in the control group. Although the amount of third-degree burns was virtually identical in the two groups, the control group required an average of 1.2 procedures per patient, whereas the impaired group required 2.1 procedures per patient. A list of possible complications or adverse reactions that could occur was used to compare the two groups. The control group averaged 1.83 complications per patient, and the impaired group averaged 3.16 complications per patient. The average length of stay in the hospital for the control group was 19 days, and the average length of stay for the impaired group was 34.1 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron transfer associated with oxygen activation in the B2 protein of ribonucleotide reductase from Escherichia coli.

Each of the two beta peptides which comprise the B2 protein of Escherichia coli ribonucleotide reductase (RRB2) possesses a nonheme dinuclear iron cluster and a tyrosine residue at position 122. The oxidized form of the protein contains all high spin ferric iron and 1.0-1.4 tyrosyl radicals per RRB2 protein. In order to define the stoichiometry of in vitro dioxygen reduction catalyzed by fully reduced RRB2 we have quantified the reactants and products in the aerobic addition of Fe(II) to metal-free RRB2apo utilizing an oxygraph to quantify oxygen consumption, electron paramagnetic resonance to measure tyrosine radical generation, and Mössbauer spectroscopy to determine the extent of iron oxidation. Our data indicate that 3.1 Fe(II) and 0.8 Tyr122 are oxidized per mol of O2 reduced. Mössbauer experiments indicate that less than 8% of the iron is bound as mononuclear high spin Fe(III). Further, the aerobic addition of substoichiometric amounts of 57Fe to RRB2apo consistently produces dinuclear clusters, rather than mononuclear Fe(III) species, providing the first direct spectroscopic evidence for the preferential formation of the dinuclear units at the active site. These stoichiometry studies were extended to include the phenylalanine mutant protein (Y122F)RRB2 and show that 3.9 mol-equivalents of Fe(II) are oxidized per mol of O2 consumed. Our stoichiometry data has led us to propose a model for dioxygen activation catalyzed by RRB2 which invokes electron transfer between iron clusters.

Enhancement of wound healing by topical treatment with epidermal growth factor.

Experimental studies in animals have demonstrated that the topical application of epidermal growth factor accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. We conducted a prospective, randomized, double-blind clinical trial using skin-graft-donor sites to determine whether epidermal growth factor would accelerate the rate of epidermal regeneration in humans. Paired donor sites were created in 12 patients who required skin grafting for either burns or reconstructive surgery. One donor site from each patient was treated topically with silver sulfadiazine cream, and one was treated with silver sulfadiazine cream containing epidermal growth factor (10 micrograms per milliliter). The donor sites were photographed daily, and healing was measured with the use of planimetric analysis. The donor sites treated with silver sulfadiazine containing epidermal growth factor had an accelerated rate of epidermal regeneration in all 12 patients as compared with that in the paired donor sites treated with silver sulfadiazine alone. Treatment with epidermal growth factor significantly decreased the average length of time to 25 percent and 50 percent healing by approximately one day and that to 75 percent and 100 percent healing by approximately 1.5 days (P less than 0.02). Histologic evaluation of punch-biopsy specimens taken from the centers of donor sites three days after the onset of healing supported these results. We conclude that epidermal growth factor accelerates the rate of healing of partial-thickness skin wounds. Further studies are required to determine the clinical importance of this finding.

Mössbauer and EPR studies of the binuclear iron center in ribonucleotide reductase from Escherichia coli. A new iron-to-protein stoichiometry.

57Fe-enriched ribonucleotide reductase subunit B2 from Escherichia coli strain N6405/pSPS2 has been characterized by Mössbauer and EPR spectroscopy in its native diferric state and in a new differous form. The native protein exhibits two Mössbauer doublets in a 1:1 ratio with parameters that are in excellent agreement with those reported for the wild-type protein (Atkin, C. L., Thelander, L., Reichard, P., and Lang, G. (1983) J. Biol. Chem. 248, 7464-7472); in addition, our studies show the absence of adventitiously bound iron. The iron content in the present samples approached 4 per B2 subunit, and the tyrosyl radical content exceeded 1 per B2 subunit. The higher values are attributed to the use of a new epsilon 280 for the protein and more efficient methods for iron extraction. We thus propose that subunit B2 has two binuclear iron clusters, each associated with its own tyrosyl radical, in contradistinction from the prevailing model. Reduction of the native protein with dithionite or reconstitution of the apoprotein with Fe(II) afforded a protein complex with Mössbauer parameters, delta EQ = 3.13 mm/s and delta = 1.26 mm/s at 4.2 K, and a low field EPR signal associated with an integer spin system. These spectral properties resemble those of methane monooxygenase in its diferrous form. Upon exposure to O2, the reduced subunit B2 readily converts to the diferric state and yields active enzyme.

Hepatitis B surface antigen associated nephrotic syndrome.

Hepatitis B virus (HBV) has been reported in association with the nephrotic syndrome from different parts of the world, but its role as a cause of the pathological findings of nephrotic syndrome is still controversial. We report seven nephrotic children with positive hepatitis B markers in which members of their families were also positive for the markers but without clinical, renal or hepatic involvement. Four showed haematuria at onset and three developed hypertension later in the course of the disease. Only two were responsive to steroid therapy. Renal biopsy was performed in four, of whom three showed membranous nephropathy and the other showed mesangioproliferative glomerulonephritis. Four patients developed end-stage renal disease. We conclude that in our environment HBV, when detected in children with nephrotic syndrome, should not be considered as a chance finding, but may have a definite role in its pathogenesis. Moreover, the prognosis of HBV-associated nephrotic syndrome appears poor.