Primary adenovirus-specific cytotoxic T lymphocyte response occurs after viral clearance and liver enzyme elevation.

The virus-specific cytotoxic T lymphocyte (CTL) response is a major obstacle to effective delivery of adenovirus gene therapy. However, its relative role in viral clearance, transgene elimination and hepatotoxicity remains unclear. In this paper, we present an analysis of viral clearance and liver toxicity in relation to the induction of the virus-specific CD8 T-cell response revealed by an MHC class I tetramer. A surprisingly high number of tetramer+ CD8 T cells were found in the liver and lung and reached peak values at days 8 and 10, respectively, post-infection. Nearly 100% of these tetramer+ CD8 T cells expressed high levels of granzyme B and IFNgamma. Remarkably, liver viral load and liver enzyme elevation peaked early, at days 2 and 4, respectively, post-infection, before the specific CTL response was detectable. After generation of CTLs, there was only minimal liver damage or further decrease in virus titer. These results indicated that the primary peak response of tetramer+ CTLs does not correlate with the elimination of adenovirus or liver cytotoxic response.

A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon.

Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8(+) T lymphocytes are discussed.

Poliovirus replicons encoding the B subunit of Helicobacter pylori urease elicit a Th1 associated immune response.

The development of a vaccine for Helicobacter pylori is a key strategy for reducing the worldwide prevalence of H. pylori infection. Although immunization with recombinant B subunit of H. pylori urease (ureB) has yielded promising results, for the most part, these studies relied on the use of strong adjuvant, cholera toxin, precluding the use in humans. Thus, the development of new vaccine strategies for H. pylori is essential. Previous studies from our laboratory have described a vaccine vector based on poliovirus in which foreign genes are substituted for the poliovirus capsid genes. The genomes encoding foreign proteins (replicons) are encapsidated into authentic poliovirions by providing the capsids in trans. To test the utility of replicons as a vaccine vector for H. pylori, a replicon was constructed which encodes ureB. Expression of ureB in cells from the replicon was demonstrated by metabolic labeling followed by immunoprecipitation with anti-urease antibodies. To investigate the immunogenicity of the replicons, mice containing the transgene for the receptor for poliovirus were immunized via the intramuscular route. Mice given three doses of replicons did not develop substantial antibodies to ureB as determined by Western blot analysis using lysates from H. pylori. In contrast, mice given two doses of replicon followed by a single injection of recombinant ureB developed serum antibodies to ureB which were predominately IgG2a. Splenic lymphocytes from mice immunized with replicons alone, or replicons plus recombinant ureB produced abundant interferon-gamma and no detectable interleukin-4 upon stimulation with recombinant ureB. These results establish that poliovirus replicons encoding H. pylori ureB are immunogenic and induce primarily a T helper 1 associated immune response.

Outcome of avulsion fractures of the ulnar base of the proximal phalanx of the thumb treated nonsurgically.

We report a retrospective study of avulsion fractures of the ulnar collateral ligament of the thumb metacarpophalangeal joint that were treated nonsurgically. The study included 30 patients who answered a questionnaire. None of the patients underwent surgery after treatment. The average follow-up interval was 3.1 years (range, 1-5.2 years). All 30 patients were satisfied with the treatment and outcome. No patient had a permanent job change or inability to participate in recreational activities. Nineteen patients had no pain on motion of the thumb. Of the 20 patients (67%) who agreed to a follow-up examination, key pinch and grip strength were not statistically different between the injured and noninjured sides. Three patients had instability on stress testing. There was a 25% nonunion rate. While not statistically significant, symptomatic patients tended to have slightly larger fragments (5.4 mm x 1.7 mm vs 3.2 mm x 1.4 mm on anterioposterior x-rays) and greater initial rotation (26 degrees vs 24 degrees) of the fracture than asymptomatic patients. Despite the nonunion rate and the patients with clinical instability, all 30 patients were satisfied with their treatment.

Subacute scaphoid fractures. A closer look at closed treatment.

Twenty-three subacute scaphoid fractures were retrospectively reviewed to determine the efficacy of nonoperative treatment. All of the patients sought medical attention between 4 weeks and 6 months after injury, and their fractures were classified according to location and stability. Nineteen fractures were observed to radiographic union or until closed treatment was abandoned; four patients were lost to followup. Nine of 10 stable subacute middle-third scaphoid fractures healed with cast immobilization in an average of 19 weeks (range, 11 to 38), and these were compared with a randomly selected group of acute middle-third fractures that healed in an average of 10 weeks (range, 6 to 13). Five of six unstable subacute middle-third fractures healed in an average of 20 weeks. One of these had a symptomatic humpback deformity treated by cheilectomy. Of three subacute proximal-third fractures, only one healed after 29 weeks of closed treatment. This study demonstrates that stable subacute middle-third scaphoid fractures will heal with cast treatment but may take twice as long to do so as stable acute middle-third fractures. Unstable subacute middle-third scaphoid fractures and subacute proximal-third fractures appear less likely to heal with closed treatment.

Bony skier's thumb injuries.

Avulsion fractures of the ulnar collateral ligament of the thumb metacarpophalangeal joint (bony skier's thumb) may result in chronic instability with pain and weakness of pinch if improperly treated. Management requires an understanding of the relevant anatomy and careful clinical examination including stress testing. Undisplaced, or minimally displaced and stable fractures are treated conservatively, whereas displaced, rotated and unstable fractures require surgical treatment.

Oligomerization of the hydrophobic heptad repeat of gp41.

The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.

Polyclonal rabbit antisera that detect the Vpr protein of SIVSM and SIVMAC on immunoblots of purified virions.

Antisera suitable for detection of SIVSM or SIVMAC Vpr proteins on Western blots of purified virions are currently not available. We have expressed the Vpr protein of SIVSMPBj1.9 in a gst-based prokaryotic expression system and used it to raise polyclonal antisera in rabbits. Two immune sera were obtained that specifically recognized both cell- and virion-associated Vpr protein on immunoblots of three different SIV isolates (SIVSMPBj1.9, SIVMACBK28, and SIVMAC239). Because Vpr is believed to play an important role in HIV/SIV replication and pathogenesis, these reagents will allow the extension of functional analyses of this protein to a broader spectrum of viruses. Both antisera and the gst-Vpr expression plasmid have been submitted to the NIAID AIDS Research and Reagent Program and are available to interested investigators.

Acute median neuropathy after wrist trauma. The role of emergent carpal tunnel release.

Ten cases of acute carpal tunnel syndrome (ACTS) and six cases of nerve contusion were identified in patients with acute median neuropathy associated with blunt wrist trauma. The patients with ACTS initially had normal sensation and subsequently developed objective sensory loss (2-point discrimination greater than 15 mm) in the median nerve distribution associated with severe wrist pain. Patients with nerve contusion injuries had immediate sensory loss and symptoms were nonprogressive. Wick catheter measurements of the carpal canal pressure were used in seven patients to help distinguish ACTS (pressure greater than 40 mm Hg) from nerve contusion. The interstitial carpal tunnel pressure was elevated an average of 52 mm Hg in four of five patients with ACTS but was normal in two patients with nerve contusion. Four of five patients who underwent carpal tunnel release within 40 hours of the onset of numbness had normal 2-point discrimination within 96 hours. The results of this study and review of the literature reflect the urgency of carpal tunnel release in ACTS. Neuropathy, secondary to nerve contusion without coexisting ACTS, may be treated initially by observation. Acute carpal tunnel syndrome must be distinguished from nerve contusion as a cause of acute posttraumatic median neuropathy.

Genetically improved potatoes: protection from damage by Colorado potato beetles.

Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.

Cytokine-specific ELISPOT assay. Single cell analysis of IL-2, IL-4 and IL-6 producing cells.

In order to assess cytokine-producing cells at the single cell level, the cytokine-specific ELISPOT assay has proven to be an important and sensitive method. The purpose of this study was to adapt this method to elucidate individual cells producing murine IL-2, IL-4 or IL-6. In order to establish these cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific cDNA transfected myeloma cell lines, e.g., X63-Ag8-653 X2, X63-Ag8-653 X4 and X63-Ag8-653 X6, respectively, were used as specific cytokine-producing cells. In the IL-2 ELISPOT assay, the coating reagent, monoclonal antibody (mAb) rat IgG2a anti-mouse IL-2 (CR #40014) was used while rabbit IgG polyclonal anti-mouse IL-2 was employed for detection of IL-2 spot forming cells (SFC). The mAbs anti-mouse IL-4, BVD4-1D11 and BVD6-24G2 were selected as capture and detection antibodies for enumeration of IL-4 SFC. For the IL-6 ELISPOT assay, anti-mouse IL-6 (MP5-20F3) mAb was used for coating and MP5-32C11 mAb was used for detection of IL-6 SFC. When IL-2 producing X63-Ag8-653 X2 cells were subjected to these three different ELISPOT assays, IL-2-specific SFC were only noted with the IL-2 ELISPOT system. In the case of IL-4 SFC, only X63-Ag8-653 X4 cells formed specific spots using the tandem of BVD4-1D11 and BVD6-24G2 mAbs. IL-6-specific spots developed in MP5-20F3 mAb pre-coated wells containing X63-Ag8-653 X6 cells, when developed with mAb anti-IL-6 (MP5-32C11). Addition of cycloheximide (50 micrograms/ml) inhibited formation of IL-2, IL-4 and IL-6 SFC by approximately 90%. When an unrelated mAb was used as detection antibody in these three different cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific SFC were not detected. Further, when concanavalin A stimulated T cells from Peyer's patch of normal mice were subjected to the respective cytokine-specific ELISPOT assay, IL-2, IL-4 and IL-6 SFC were enumerated. These results have shown that cytokine-specific IL-2, IL-4 and IL-6 ELISPOT assays have now been established and will allow analysis of the frequency of cytokine-secreting cells at the single cell level.

Digital pacinian corpuscle neuroma eroding bone: a case report.

A case of digital pacinian corpuscle neuroma leading to erosive changes in the adjacent proximal phalanx is reported. Characteristics and previous reports of this relatively uncommon hand tumor are discussed.

Cubital tunnel syndrome.

Cubital tunnel syndrome is the second most common compressive neuropathy of the upper extremity. Key factors in the history, physical, and differential are outlined to assist the clinician in making an accurate diagnosis. Nonoperative measures and surgical options are reviewed, with medial epicondylectomy being the authors' preferred operative technique.

Cloning of a Serratia marcescens Gene Encoding Chitinase.

Serratia marcescens, a chitinase-producing microorganism, was shown to produce five unique chitinolytic proteins with subunit molecular masses of 21, 36, 48, 52, and 57 kilodaltons. A cosmid library of S. marcescens DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in Escherichia coli for clones capable of degrading chitin. A total of four independent clones (22- to 27-kilobase inserts) were isolated, characterized by restriction endonuclease digestion, and shown to share a common 9.5-kilobase EcoR1 fragment apparently encoding the same 57-kilodalton chitinase, the most abundant chitinase produced by S. marcescens. Chitinase expression from these constructs in both E. coli and Pseudomonas fluorescens 701E1 is apparently driven by an S. marcescens promoter. The significantly higher chitinase levels produced in E. coli relative to those in P. fluorescens 701E1 suggest that E. coli may recognize this promoter sequence more efficiently than P. fluorescens.