T-cell response to adenovirus hexon and DNA-binding protein in mice.

The successful development of adenovirus vectors for vaccines and gene therapy will require a better understanding of the host immune response. Using the ELISPOT assay to measure IFN-gamma-secreting CD8(+) cells, we identify immunodominant epitopes of the adenovirus hexon and DNA-binding protein in BALB/c and C57BL/6 mice. The T-cell response to the intramuscular administration of adenovirus serotype 5 peaks within a few weeks and gradually declines but is still detectable after 12 weeks. A second administration did not substantially increase the number of reactive T cells. The CD8(+) T-cell response was also similar between wild type and E1-deleted adenovirus. When B-cell-deficient mice were injected with adenovirus encoding the gene for secreted alkaline phosphatase, sera phosphatase activity was reduced more quickly in mice pre-exposed to adenovirus. These results add to the evidence that cell-mediated immunity is a substantial barrier to therapeutic adenoviral vectors and provide more quantitative tools to measure cellular immune responses to adenovirus.

Chemical peels.

For many decades, chemical peeling has played an important role in facial rejuvenation. These procedures are safe and effective in the management of photoaging, scarring, pigmentary dyschromias, and in the destruction of superficial skin lesions. Chemical peels are classified into superficial, medium, and deep according to their level of injury to the skin. It is the responsibility of the surgeon to assist the patient in choosing the resurfacing procedure that will most effectively satisfy his or her goals while maintaining a high margin of safety. With the current availability of various microdermabrasion and laser systems for facial rejuvenation, chemical peeling continues to be an integral part of a facial rejuvenation program because of its popularity with patients and minimal costs to the physician.

Deep dermatophytosis: report of 2 cases and review of the literature.

Skin infections due to dermatophytes are common and generally associated with a low degree of morbidity in normal hosts. Rare cases have been reported in which the dermatophyte invaded the deep dermis, subcutis, or even internal organs. Two patients, each of whom had clinical and histological findings of a deep or locally invasive dermatophyte infection, are described. This condition typically presents as a nodular eruption that is characterized histologically by suppurative granulomatous inflammation and deposition of organisms in the reticular dermis. Recognition of the potential of dermatophytes for local invasion in susceptible hosts will help ensure proper diagnosis and timely intervention in these cases.

HIV lipodystrophy: review of the syndrome and report of a case treated with liposuction.

BACKGROUND: A syndrome characterized by loss of fat on the face and limbs, localized fatty deposits on the trunk, and metabolic disturbances is becoming increasingly recognized in the human immunodeficiency virus (HIV) patient population. OBJECTIVE: To increase awareness of this syndrome among dermatologists and dermatologic surgeons and to review its various treatment options, including liposuction. METHODS: We present a patient with HIV lipodystrophy syndrome who underwent tumescent liposuction. We also describe our experience with liposuction in the management of this condition and review the treatment options that have been proposed in the literature. RESULTS: In the medical management of HIV lipodystrophy, various agents have been utilized but most have yielded disappointing results. Preliminary evidence on the use of tumescent liposuction in these patients suggests that significant improvement in the cosmetic disfigurement can be achieved. CONCLUSION: This syndrome is common among HIV-infected patients and remains difficult to treat. Although medical therapy may be preferable in most patients, liposuction represents a viable option in selected individuals.

The glucagonoma syndrome: a review of its features and discussion of new perspectives.

Glucagonoma syndrome is a paraneoplastic phenomenon characterized by an islet alpha-cell pancreatic tumor, necrolytic migratory erythema, diabetes mellitus, weight loss, anemia, stomatitis, thromboembolism, and gastrointestinal and neuropsychiatric disturbances. These clinical findings in association with hyperglucagonemia and demonstrable pancreatic tumor establish the diagnosis. Glucagon itself is responsible for most of the observed signs and symptoms, and its induction of hypoaminoacidemia is thought to lead to necrolytic migratory erythema. Liver disease and fatty acid and zinc deficiency states may also contribute to the pathogenesis of the eruption in some cases. Most patients are diagnosed too late in the clinical course for cure, but successful palliation of symptomatology can usually be achieved with surgical and medical intervention. This paper reviews the glucagonoma syndrome, paying particular attention to its cutaneous features, and provides new perspectives in our current understanding of this phenomenon.

Antigen levels and antibody titers after DNA vaccination.

DNA vaccination generates strong cellular and humoral immunity in animal models. The mechanisms by which plasmid DNA uptake and expression after intramuscular injection lead to immune responses are not well understood. In particular, the importance of antigen expression levels on subsequent antibody immune responses has not been established. We found that a chemiluminescent assay for alkaline phosphatase allows measurement of antigen levels of secreted alkaline phosphatase (SEAP) in vivo after intramuscular injection of a wide range of plasmid doses. The mice produced antibodies to the alkaline phosphatase reporter gene and both antigen levels and antibody titers were measured over time. We found that the correlation between initial antigen level and antibody response was high (r = 0.74, p < 0.001) and remained high even after accounting for the dose of plasmid injected (r = 0.61, p < 0.001). The correlation between DNA dose and antibody titer was statistically significant (r = 0.53, p < 0.001) but was reduced to almost zero after we accounted for initial antigen levels.

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

Pilomatrix dysplasia in an immunosuppressed patient.

Immunosuppressive drugs have been used for many years in the prevention of graft failure in transplant recipients. Although they improve morbidity and mortality after transplantation, these medications carry a significant risk of adverse mucocutaneous and systemic effects. We describe a patient receiving 4 immunosuppressive drugs who experienced persistent facial dysmorphism along with striking follicular disturbances. On histopathologic examination, the follicular structures were dilated and hyperplastic with a peculiar dysplasia of the pilar matrix. Based on a review of the clinical, microscopic, and investigational findings of the skin previously reported in association with her immunosuppressive drugs, we conclude that cyclosporine was the most likely causative agent. Moreover, hypertrichosis, dysmorphic facies, and tissue hyperplasia have all been observed in patients during cyclosporine administration.

The "facelift" flap for reconstruction of cheek, lateral orbit, and temple defects.

BACKGROUND: Large defects of the cheek, lateral orbit, zygomatic arch, or the lower temple pose challenges for reconstruction. These defects can be elegantly reconstructed using the "facelift" flap. OBJECTIVE: The facelift flap is a large advancement flap with a rotational component based on rhytidectomy principles. METHODS: Redundant skin from the lower cheek is used as the donor tissue, which is advanced cephalad and posteriorly. Flap design varies slightly for men and women depending on characteristics of the external ear and ear lobe as well as the position and density of the preauricular hairline. Extensive undermining is critical to reduce tension on the flap and allow for complete closure. Traction provided by an assistant aids in the undermining. Specialized instruments are helpful when performing this flap. Rhytidectomy scissors, multipronged skin rakes, hand-held fiberoptic lighted retractor, and insulated forceps are particularly useful. Correct trimming of the flap and ear lobe placement without tension on the lobe are essential for a good cosmetic result. A large standing cone is excised retroauricularly such that the scar is hidden primarily behind the ear. RESULTS AND CONCLUSION: The facelift flap gives superior and elegant results for reconstruction of large cutaneous defects involving the cheek, lateral orbit, zygomatic arch, and lower temple.

Propylthiouracil hypersensitivity: report of two patients with vasculitis and review of the literature.

Two patients with a hypersensitivity vasculitis in association with propylthiouracil (PTU) administration are described. Although both patients presented with a cutaneous eruption, our first patient suffered severe systemic manifestations and the second patient's involvement was primarily limited to the skin. Patients with a vascular hypersensitivity reaction to PTU typically present with constitutional symptoms, acral purpuric skin lesions, and variable involvement of multiple organ systems. The reaction is treated by urgent withdrawal of PTU and implementation of supportive measures and immunosuppressive agents, as necessary. Prompt recognition of this condition and initiation of appropriate therapy lead to complete recovery in most cases.

Enhancement of DNA vaccine potency using conventional aluminum adjuvants.

The immunogenicity and protective efficacy of DNA vaccines have been amply demonstrated in numerous animal models of infectious disease. However, the feasibility of DNA vaccines for human use is not yet known. In order to investigate potential means of increasing the potency of DNA vaccines, conventional adjuvants such as aluminum salts were tested. Coadministration of these adjuvants with DNA vaccines substantially enhanced the ability of these vaccines to induce antibody responses up to 100-fold in mice and guinea pigs, and 5-10-fold in non-human primates. Effective formulations had no demonstrable effect on the levels of antigen expression in situ and consisted of adjuvants that did not form complexes with the plasmid DNA; rather they exerted their effects on antigen after expression in situ. Therefore, the potency of DNA vaccines both in laboratory rodents and in non-human primates can be substantially increased by simple formulation with conventional aluminum adjuvants.

Evaluation of activated protein C on canine infarct size in a nonthrombotic model of myocardial reperfusion injury.

Myocardial infarct size has been measured after 1 hr of mechanical occlusion of the circumflex coronary artery and 5 hr of reperfusion in control dogs infused with saline, and in dogs infused with activated protein C (aPC) (1mg/kg/hr i.v.). Infusion of aPC during reperfusion produced a sustained doubling of activated partial thromboplastin time and no change in thrombin time at a final plasma parent drug concentration of 1.25 +/- 0.11 mug/ml. aPC infusion did not alter systolic arterial pressure, cardiac rate or the rate pressure product when compared to time-related alterations observed in control dogs. ST-segment deviation and the intensity and duration of cardiac arrhythmias associated with reperfusion of ischemic myocardium also were similar between groups. Resultant infarct sizes were 34.8 +/- 3.9 and 33.2 +/- 6.2% of the left ventricular mass placed at risk of necrosis in control and aPC-treated dogs. respectively. aPC infusion was associated with a small reduction in leukocytosis in response to myocardial ischemic injury, but did not alter the localization of leukocytes within ischemic and infarcted myocardium. In vitro concentrations of aPC (0.3, 1 and 3 mug/ml), comparable to the plasma concentration that inhibited blood coagulation in dogs, did not alter superoxide production or CD11b/CD18-mediated adhesion of chemotactic factor f-Met-Leu-Phe-stimulated neutrophils. Present data indicate that aPC lacks cardioprotectant activity at an infusion rate inhibiting coagulation. Apart from inhibition of thrombin generation, no evidence of an anti-inflammatory effect of aPC was observed.

Nucleoside triples from the group I intron.

Oligonucleotides modeled on a proposed base-triple domain in the P4/P6 region of the self-splicing group I intron have been characterized by NMR. The NMR data indicate that single-stranded nucleotides in this domain are in the minor groove of an adjacent helix within hydrogen bonding distance of 2'-hydroxyl groups in an interaction we term a nucleoside triple. Oligonucleotides containing the two most frequently occurring sequences among group I introns in the P4/P6 region formed nucleoside triples in the minor groove, whereas oligonucleotides containing sequences which are not conserved did not form triples. Surprisingly, the structures of the nucleoside triples in the oligonucleotides containing the two most frequently occurring sequences are different. If this difference were maintained in the context of the whole intron, it would suggest that the triples are not directly involved in catalysis, but rather that the nucleoside triples function by aligning the helical domains within the catalytic core of the intron.

Poly(rA) binds poly(rG).poly(rC) to form a triple helix.

Poly(rA) binds poly(rG).poly(rC) to form a triple helix. Evidence for this structure includes ultraviolet absorbance mixing curves and melting curves, and circular dichroism spectroscopy. The formation of the triple helix depends on the length of the poly(rC) strand. Triple helix forms when the average length is around 100 nucleotides but does not form when the average length is about 500 nucleotides.

A base-triple structural domain in RNA.

An oligonucleotide modeled on a proposed base-triple domain of the Tetrahymena group I intron has been characterized by NMR. The oligonucleotide contains two double-helix regions with adjacent single-stranded nucleotides. The NMR data show that the two helices stack coaxially, although the rotation between the two helices is approximately twice as large as the rotation between normal base pairs. The rotation between the two helices allows the single-stranded nucleotides to form U.U.G and A.G.C base triples in the minor groove. The A.G.C base triple contains a hydrogen bond between the adenine N1 and a 2'-hydroxyl in the minor groove of the G.C pair. A similar hydrogen bond between an adenine and a 2'-hydroxyl in transfer RNA suggests that this could be a recurring tertiary interaction in RNA.

Synthesis and purification of large amounts of RNA oligonucleotides.

Biophysical studies of RNA oligonucleotides require milligram amounts of RNA of specific length and sequence. Transcription from synthetic DNA templates using T7 RNA polymerase is a convenient method for synthesis of RNA oligonucleotides ranging in size from 9 to about 45 nucleotides. Here we present methods that make the large-scale synthesis of RNA oligonucleotides practical. This paper describes a rapid method for isolating T7 RNA polymerase free from RNases for use in transcription reactions. Protocols are also described for purification of the desired RNA oligonucleotide from the other products of transcription.