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The dynamics of photoluminescent response during optical degradation of MEH-PPV in films and chloroform solutions were investigated using various spectroscopic techniques (PL, UV-Vis, FTIR, and XRF). A significant blue shift in photoluminescence (PL) from MEH-PPV films during degradation indicated strong interchain exciton migration and a different photochemical reaction from that of solution. Two distinct wavelength-dependent dynamic PL responses were observed in MEH-PPV, and were attributed to photoactivation in the long side chain and polymer backbone. It was verified by a control experiment with BDMO-PPV which has symmetric side chains. The dynamic PL results indicate that the degradation process involves at least two-step photochemical reactions in the side chain and backbone. We also found that chlorine showed no effect on the optical degradation process.

Abstract Background Streptococcus pneumoniae (the pneumococcus) is the leading cause of otitis media, community-acquired pneumonia (CAP), sepsis, and meningitis. It is now evident that S. pneumoniae forms biofilms during nasopharyngeal colonization; the former which facilitates persistence, the latter, a prerequisite for subsequent development of invasive disease. Proteomic evaluation of S. pneumoniae suggests the antigen profile available for host-recognition is altered as a consequence of biofilm growth. This has potentially meaningful implications in regards to adaptive immunity and protection from disseminated disease. We therefore examined the antigen profile of biofilm and planktonic pneumococcal cell lysates, tested their reactivity with human convalescent sera and that generated against biofilm pneumococci, and examined whether immunization with biofilm pneumococci protected mice against infectious challenge. Results Biofilm pneumococci have dramatically altered protein profiles versus their planktonic counterparts. During invasive disease the humoral immune response is skewed towards the planktonic protein profile. Immunization with biofilm bacteria does not elicit a strong-cross-reactive humoral response against planktonic bacteria nor confer resistance against challenge with a virulent isolate from another serotype. We identified numerous proteins, including Pneumococcal serine-rich repeat protein (PsrP), which may serve as a protective antigens against both colonization and invasive disease. Conclusion Differential protein production by planktonic and biofilm pneumococci provides a potential explanation for why individuals remain susceptible to invasive disease despite previous colonization events. These findings also strongly suggest that differential protein production during colonization and disease be considered during the selection of antigens for any future protein vaccine.

The objective of this study was to determine the pharmacokinetics of two orally administered doses of tramadol (1 mg/kg and 5 mg/kg) and its metabolite, O-desmethyltramadol (M1) in giant tortoises ( Chelonoidis vandenburghi , Chelonoidis vicina ). Eleven giant tortoises ( C. vandenburghi , C. vicina ) received two randomly assigned, oral doses of tramadol (either 1 mg/kg or 5 mg/kg), with a washout period of 3 wk between each dose. The half-life (t½) of orally administered tramadol at 1 mg/kg and 5 mg/kg was 11.9 ± 4.6 h and 13.2 ± 6.1 h, respectively. After oral administration of tramadol at 1 mg/kg and 5 mg/kg, the maximum concentration (C max ) was 125 ± 69 ng/ml and 518 ± 411 ng/ml, respectively. There were not enough data points to determine pharmacokinetic (PK) parameters for the M1 metabolite from either dose. Tramadol administered orally to giant tortoises at both doses provided measurable plasma concentrations of tramadol for approximately 48 h with occasional transient sedation. Oral tramadol at 5 mg/kg, on average, achieves concentrations of >100 ng/ml, the reported human therapeutic threshold, for 24 h. Based on the low levels of M1 seen in this study, M1 may not be a major metabolite in this taxon.

This case series describes six confirmed cases of mycotic encephalitis and/or mycotic pneumonia in southern pudu ( Pudu puda ). One case involved a 10.5-yr-old intact female that presented with an inability to stand, eventually progressing to grand mal seizures. Magnetic resonance imaging showed a lesion within the cerebellar vermis with edema causing cerebellar herniation. The animal was euthanized based on a grave prognosis. Gross and histologic examination revealed primary central nervous system phaeohyphomycosis. Curvularia spicifera was sequenced from the cerebellar tissue. This is the first time this fungus has been reported as a primary central nervous system infection in an artiodactyl species. The remaining five cases occurred in neonates between 17 and 67 days old. Clinical signs varied widely, including facial swelling, weakness, posterior paresis, and sudden death. Antifungal therapy was initiated in three neonatal animals but was unsuccessful in each case. All neonates had active mycotic pneumonia caused by Aspergillus fumigatus or Mucor spp. at time of death; four of these animals also had disseminated disease that caused mycotic encephalitis. This case series indicates that fungal disease should be included in the differential diagnosis list of any pudu presenting for neurologic or respiratory clinical signs.

Kidney disease is common among captive cheetahs ( Acinonyx jubatus). Serum creatinine is the most common measurement to estimate glomerular filtration rate (GFR) because of the ease of its clinical use, but it is a crude estimate that only increases after significant disease is already present and is affected by extrarenal factors. Symmetric dimethylarginine (SDMA) is a renal biomarker in humans, dogs, and cats that correlates with serum creatinine and GFR and appears to be an earlier and more specific biomarker for kidney disease. Ninety-two banked serum samples from 11 cheetahs housed at the Oklahoma City Zoo from 1992 to 2012 were retrospectively analyzed. Histopathology results were available for 10/11 cheetahs, and all 10 had histologic renal lesions. General categories of renal lesions included glomerulosclerosis (7/10; 70%), amyloidosis (7/10; 70%), inflammatory (9/10; 90%), and oxalate nephrosis (2/10; 20%). SDMA immunoassay and mass spectrometry were measured for validation and compared with creatinine to assess for correlation. Serum creatinine concentrations were determined by enzymatic colorimetric methods. SDMA immunoassay was validated in cheetahs and correlated well with serum creatinine ( R 2 =0.687; P < 0.0001). SDMA and serum creatinine measured from freeze-thawed stored samples show high correlation in individual cheetahs ( R 2 = 0.972; P < 0.0001). These data support that SDMA could be a promising renal biomarker in cheetahs. Further research is warranted to investigate whether SDMA might be an earlier indicator of kidney disease in cheetahs and whether this assay can be extended to other nondomestic carnivores.

A 26-year-old, female hyacinth macaw (Anodorhynchus hyacinthinus) was presented for ophthalmology consultation for a history of a chronic, nonhealing corneal ulcer of the right eye. On examination, a 5 × 4-mm axial, superficial corneal ulcer with loose epithelial edges was found. During multiple recheck examinations over 4 months, the ulcer was treated by debridement with a sterile cotton-tipped applicator, diamond burr debridement, diamond burr debridement with cyanoacrylate tissue adhesive, and grid keratotomy with cyanoacrylate tissue adhesive, all performed under anesthesia with eventual resolution of the corneal ulcer.

A 5-mo-old female Rothschild's giraffe ( Giraffa camelopardalis rothschildi) presented for regurgitation. Esophagoscopy at 24 wk of age revealed a markedly dilated cranial esophagus with a tight stricture at the level of the heart base consistent with a vascular ring anomaly. Surgical exploration confirmed persistent right fourth aortic arch with ductus originating from left subclavian artery at its junction with the aorta and left subclavian artery. The patent ductus arteriosus was surgically ligated. The procedure was complicated by limited surgical access and vascular friability resulting in uncontrollable hemorrhage, and the animal was euthanatized. The animal's large size and unique shape precluded preoperative examination by computed tomography. Surgical accessibility was poor because cranial retraction of the thoracic limb was limited. Histology revealed focal degeneration of the aorta and subclavian artery and muscular degeneration of the esophagus. Degeneration was attributed to local hypoxia from compression by the vascular structure as the animal grew.

A 62-yr-old male Southern Isabela giant tortoise (Chelonoidis vicina) had a 1-yr history of chronic, reoccurring pododermatitis on the palmar surface of the left forelimb. Aggressive wound management was instituted and included surgical debridement, vacuum-assisted closure, and orthotic boot support during healing. A custom fabricated, carbon fiber clamshell Charcot Restraint Orthotic Walker walking boot was utilized to reduce focal pressure over the wound during weight bearing and promote a more normal gait. Distal padding was used to distribute pressure on the palmar surface of the left forelimb, with a focal depression in the padding preventing pressure directly over the wound. The design and trim lines were adjusted to allow shoulder and elbow motion without impingement. The clamshell design allowed relatively easy removal for wound inspection and dressing changes. The wound ultimately resolved after 9 wk of management with the orthotic boot, with no reoccurrence over the next 3 yr.

Short-term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Stereotaxy is a neurosurgical technique that can take several hours to reach a specific target, typically utilizing a mechanical frame and guided by preoperative imaging. An error in any one of the numerous steps or deviations of the target anatomy from the preoperative plan such as brain shift (up to mm), may affect the targeting accuracy and thus the treatment effectiveness. Moreover, because the procedure is typically performed through a small burr hole opening in the skull that prevents tissue visualization, the intervention is basically “blind” for the operator with limited means of intraoperative confirmation that may result in reduced accuracy and safety. The presented system is intended to address the clinical needs for enhanced efficiency, accuracy, and safety of image-guided stereotactic neurosurgery for deep brain stimulation lead placement. The study describes a magnetic resonance imaging (MRI)-guided, robotically actuated stereotactic neural intervention system for deep brain stimulation procedure, which offers the potential of reducing procedure duration while improving targeting accuracy and enhancing safety. This is achieved through simultaneous robotic manipulation of the instrument and interactively updated in situ MRI guidance that enables visualization of the anatomy and interventional instrument. During simultaneous actuation and imaging, the system has demonstrated less than 15% signal-to-noise ratio variation and less than 0.20 geometric distortion artifact without affecting the imaging usability to visualize and guide the procedure. Optical tracking and MRI phantom experiments streamline the clinical workflow of the prototype system, corroborating targeting accuracy with three-ax- s root mean square error 1.38 ± 0.45 mm in tip position and 2.03 ± 0.58° in insertion angle.

Ocular disease appears to be a common issue in anurans. Intraocular pressures were measured for six species of free-ranging anurans in central Oklahoma. No significant differences were identified between left or right eyes. There was a significant negative relationship between the weight of the anuran and intraocular pressure. The intraocular pressure range for the six species was 3-10 mm Hg. Tonometry values in anurans are, to the authors' knowledge, previously unreported and this study provides initial information on intraocular pressure measurement in anurans.

This report describes two cases of retroperitoneal abscesses in female western lowland gorillas (Gorilla gorilla gorilla). Clinical symptoms included perivulvar discharge, lameness, hindlimb paresis, and general malaise. Retroperitoneal abscesses should be considered as part of a complete differential list in female gorillas with similar clinical signs.

Dexmedetomidine, a highly selective alpha-2 adrenergic agonist and dextrorotary enantiomer of medetomidine, was combined with ketamine and used to immobilize 14 free-ranging Choloepus hoffmanni (Hoffmann's two-toed sloths) and 11 Bradypus variegatus (brown-throated three-toed sloths) in Upala, Costa Rica. Following intramuscular injection of ketamine (2.1 mg/kg) and dexmedetomidine (11 microg/kg), heart rate, respiratory rate, and indirect systolic blood pressure were measured every 5 min for a total of 25 min. An iStat (CG8+) was used to evaluate serum biochemical and hematologic values during anesthesia. After 30 min of anesthesia, atipamezole (0.13 mg/kg) was administered intramuscularly, which resulted in rapid and smooth recoveries. Mean heart rate and respiratory rate remained unchanged in both C. hoffmanni and B. variegatus over time. Progressive decreases in mean indirect systolic blood pressure were documented in both species. Results of this study suggest a combination of dexmedetomidne and ketamine is a safe and effective anesthetic protocol for use in free-ranging C. hoffmanni and B. variegatus. Similar to other alpha-2 adrenergic agonist-based immobilization protocols, close monitoring of cardiovascular and respiratory parameters are recommended. This study also provides serum biochemical and hematologic data in free-ranging C. hoffmanni and B. variegatus.

Objective: To compare pharmacokinetics after IV, IM, and oral administration of a single dose of meloxicam to Hispaniolan Amazon parrots (Amazona ventralis)., Animals: 11 healthy parrots., Procedures: Cohorts of 8 of the 11 birds comprised 3 experimental groups for a crossover study. Pharmacokinetics were determined from plasma concentrations measured via high-performance liquid chromatography after IV, IM, and oral administration of meloxicam at a dose of 1 mg/kg., Results: Initial mean ± SD plasma concentration of 17.3 ± 9.0 μg/mL was measured 5 minutes after IV administration, whereas peak mean concentration was 9.3 ± 1.8 μg/mL 15 minutes after IM administration. At 12 hours after administration, mean plasma concentrations for IV (3.7 ± 2.5 μg/mL) and IM (3.5 ± 2.2 μg/mL) administration were similar. Peak mean plasma concentration (3.5 ± 1.2 μg/mL) was detected 6 hours after oral administration. Absolute systemic bioavailability of meloxicam after IM administration was 100% but was lower after oral administration (range, 49% to 75%). Elimination half-lives after IV, IM, and oral administration were similar (15.9 ± 4.4 hours, 15.1 ± 7.7 hours, and 15.8 ± 8.6 hours, respectively)., Conclusions and Clinical Relevance: Pharmacokinetic data may provide useful information for use of meloxicam in Hispaniolan Amazon parrots. A mean plasma concentration of 3.5 μg/mL would be expected to provide analgesia in Hispaniolan Amazon parrots; however, individual variation may result in some birds having low plasma meloxicam concentrations after IV, IM, or oral administration. After oral administration, meloxicam concentration slowly reached the target plasma concentration, but that concentration was not sustained in most birds.

This paper describes a modular design approach for robotic surgical manipulator under magnetic resonance imaging (MRI) guidance. The proposed manipulator provides 2 degree of freedom (DOF) Cartesian motion and 2-DOF pitch and yaw motion. Primarily built up with dielectric materials, it utilizes parallel mechanism and is compact in size to fit into the limited space of close-bore MRI scanner. It is ideal for needle based surgical procedures which usually require positioning and orientation control for accurate imaging plane alignment. Specifically, this mechanism is easily reconfigurable to over constrained manipulator structure which provides 2-DOF Cartesian motion by simple structure modification. This modular manipulator integrated with different end-effector modules is investigated for prostate brachytherapy and neurosurgery applications as preliminary evaluation.

This paper presents the design of a magnetic resonance imaging (MRI) compatible needle placement system actuated by piezoelectric actuators for prostate brachytherapy and biopsy. An MRI-compatible modular 3 degree-of-freedom (DOF) needle driver module coupled with a 3-DOF x-y-z stage is proposed as a slave robot to precisely deliver radioactive brachytherapy seeds under interactive MRI guidance. The needle driver module provides for needle cannula rotation, needle insertion and cannula retraction to enable the brachytherapy procedure with the preloaded needles. The device mimics the manual physician gesture by two point grasping (hub and base) and provides direct force measurement of needle insertion force by fiber optic force sensors. The fabricated prototype is presented and an experiment with phantom trials in 3T MRI is analyzed to demonstrate the system compatibility.

OBJECTIVE-To evaluate the analgesic efficacy of meloxicam in parrots with experimentally induced arthritis, with extent of weight bearing and rotational perch walking used as outcome measures. ANIMALS-15 adult Hispaniolan parrots (Amazona ventralis). PROCEDURES-Arthritis was experimentally induced via intra-articular injection of microcrystalline sodium urate suspension (MSU) into 1 intertarsal joint. Parrots were treated in a crossover design. Five treatments were compared as follows: meloxicam (4 dosages) at 0.05, 0.1, 0.5, and 1.0 mg/kg (IM, q 12 h, 3 times) and 0.03 mL of saline (0.9% NaCl) solution (IM, q 12 h, 3 times). The first treatment was given 6 hours following MSU administration. Lameness was assessed by use of a biomechanical perch to record weight-bearing load and a rotational perch to determine dexterity. Feces were collected to assay for occult blood. RESULTS-Parrots treated with meloxicam at 1.0 mg/kg had significantly better return to normal (baseline) weight bearing on the arthritic pelvic limb, compared with control parrots or parrots treated with meloxicam at 0.05, 0.1, and 0.5 mg/kg. All fecal samples collected from parrots following induction of arthritis and treatment with meloxicam had negative results for occult blood. CONCLUSIONS AND CLINICAL RELEVANCE-Meloxicam administered at 1.0 mg/kg, IM, every 12 hours effectively relieved arthritic pain in parrots.

The work presented in this paper has been performed in furtherance of developing an MRI-compatible surgical robotic system, specifically targeting the neural intervention procedure for the treatment of Parkinson's Syndrome known as deep brain stimulation (DBS). In this paper we discuss the construction and testing of the MR-compatible controller, sensors and actuators, and the compatibility testing we have done to validate the success of our efforts in eliminating signal interference. Our robotic system was tested on a Phillips Achieva 3 Tesla MRI machine under diagnostic T1 and T2, high speed FGRE and functional EPI imaging protocols. It has been shown to operate without introducing any statistically significant degradation in image quality. We have shown that the creation of an MR-compatible electronically controlled closed-loop robotic actuation system and linkage mechanism can be created successfully within a standard high-field diagnostic magnet with insignificant levels of signal interference.

Reintroduction of endangered Whooping Cranes (Grus americana) in eastern North America has successfully established a migratory population between Wisconsin and Florida. Eighty birds (47 males, 33 females) were released between 2001 and 2006, and all birds were tracked following release with satellite and/or VHF monitoring devices. By the end of 2006, 17 deaths (12 males, five females) were recorded from this population. Postmortem findings and field data were evaluated for each bird to determine the cause of death. Causes included predation (n=8, 47%), trauma (n=2, 12%), and degenerative disease (n=1, 6%); the cause of death was undetermined for 35% (n=6) of the birds. Based on physical evidence, the primary predator of the birds was the bobcat (Lynx rufus). Limited roosting habitat availability or bird behavior were likely prime factors in the occurrence of predation. Traumatic injuries and mortality were caused by gunshot, electrical utility lines, and an unknown source. The lone case of degenerative disease was due to chronic exertional myopathy associated with translocation. Available postmortem testing did not indicate the presence of infectious disease in this limited sample.

A 1.5-year-old male Moluccan cockatoo (Cacatua moluccensis) presented to Kansas State University Veterinary Medical Teaching Hospital with a 4-month history of lethargy and weakness. Hematologic and radiographic diagnostic testing revealed profound leukocytosis and coelomic and pulmonary granulomatous masses of unknown origin. The bird died during laparoscopic evaluation under general anesthesia. Necropsy revealed multiple pulmonary and hepatic soft-tissue nodules and an intracoelomic mass over the left kidney communicating with external subcutaneous masses and a pericloacal mass of similar gross appearance. Histopathologic findings identified a severe, disseminated, inflammatory infiltration of multiple tissues and multiple granulomas containing bizarre multinucleated cells. No causative agent of this granulomatous disease was identified. To our knowledge, this is the first report of systemic atypical granulomatous disease in Moluccan cockatoo. Traditional causes of granulomatous disease include mycotic disease, bacterial (ie, Mycobacterium) disease, and neoplasia. Attempts to identify an causative agent or neoplasia were unsuccessful. A retrospective review of pathology records revealed 2 additional cases with identical pathologic lesions. All 3 cases occurred in young Moluccan cockatoos and are assumed to be a disease of unknown origin that could be unique to this species.

Effective screening of new vaccines and immunotherapeutics requires assay methods that can provide quantitative measurement of cellular immune responses. The enzyme-linked immunospot (ELISPOT) is a sensitive technique for the detection of cytokine-producing cells at the single cell level. This assay is rapid and reproducible and permits the direct enumeration of low-frequency antigen-specific T-cells. This protocol describes in detail an interferon (IFN)-gamma ELISPOT method for measuring antigen-specific murine CD8+ T-cells. Spleen cells from specific cytotoxic T-lymphocyte (CTL) peptide-primed mice are used source of CD8+ T-cells to demonstrate the utility of this technique. The assay procedure is facilitated by the use of a ready-to-use IFN-gamma ELISPOT assay kit and it can be adapted to other model systems in which CD8+ T-cell responses are monitored.

Objective: The purpose of this study was to evaluate the safety of the administration of a bacterial expression plasmid encoding a 13 amino acid sequence that is highly homologous with human papillomavirus E7 within poly (lactide-co-glycolide) microparticles (ZYC101) in women with HLA A2+ antigen and persistent cervical intraepithelial neoplasia grade 2/3 and human papillomavirus 16., Study Design: Fifteen women entered an institutional review board-approved dose-escalating phase I study with the use of three levels of blood monitoring and urine studies, Papanicolaou tests, and colposcopy. Escalation required no serious adverse events. Immunologic responses were evaluated in peripheral blood with the use of human papillomavirus peptide-stimulated interferon gamma enzyme-linked immunosorbent assay for T-cell reactivity. In cervical secretions, immunoglobulin A anti-human papillomavirus 16 E2 concentrations were measured. Three doses every 3 weeks were followed 4 weeks later by surgical excision., Results: No serious adverse events occurred. Five women had complete histologic responses; 11 women had human papillomavirus-specific T-cell responses. Four of five complete histologic responses developed immunoglobulin A anti-E2-specific antibody., Conclusion: ZYC101 warrants further investigation because of a 33% complete histologic responses, a 73% immunologic response, and no serious adverse events.

Positive practice overcorrection (PPOC) has long played a significant role in the behavioral treatment of serious self-stimulatory behavior. Three experiments comparing the effectiveness of 30-second, 2-minute, and 8-minute PPOC on reduction of stereotypic hand behavior of adults with severe to profound developmental disabilities were conducted to resolve inconsistencies in previously reported findings concerning the role of PPOC duration in response suppression. Experiment 1, which used an alternating treatments--multiple baseline design, suggested that the different durations were equally effective in reducing the stereotypic behaviors to near-zero levels. Experiment 2, which used a reversal design, supported the findings of Experiment 1. Experiment 3, which used a reversal design to test the shortest and longest durations, generally confirmed the results of the first two experiments. This study therefore failed to support the oft-claimed superiority of long-duration PPOC. The possible factors underlying these findings and their implications for future research and practice are discussed.

The relationship between the primary effector CTL response to viral infection and the subsequent pool of memory CTL precursors (CTLp) is poorly understood. Here, we have analyzed the induction of both effector CTL and memory CTLp to dominant and subdominant epitopes following Sendai virus infection of C57BL/6 mice. A single peptide derived from the Sendai virus nucleoprotein (NP(324-332)) binds to both H-2 Kb and Db MHC class I molecules, generating both immunodominant (NP(324-332)/Kb) and subdominant (NP(324-332)/Db) epitopes. Following intranasal Sendai virus infection, NP(324-332)/Kb-specific CTL dominated the primary effector CTL response in the lung and were present at high frequency in the memory CTLp pool. In contrast, NP(324-332)/Db-specific CTL were not a detectable component of the effector response to primary Sendai virus infection. However, memory CTLp specific for this subdominant epitope were induced at frequencies approaching those of CTLp specific for the immunodominant epitope. These data indicate that memory CTLp specific for subdominant epitopes can be primed by Sendai virus infection in the absence of a detectable effector response. To determine whether CTLp memory to subdominant epitopes is functional in the context of Sendai virus infection, memory CTLp specific for a subdominant epitope were selectively primed by vaccination. These cells dominated the subsequent effector CTL response to Sendai virus infection, demonstrating that memory CTLp primed against subdominant epitopes can participate in an immune response and effectively compete with T cells specific for immunodominant epitopes. These data have implications for the development of vaccines designed to emphasize cellular immunity.

Recent studies have shown that only a subset of major histocompatibility complex (MHC) class II molecules are able to present bacterial superantigens to T cells, leading to the suggestion that class-II associated peptides may influence superantigen presentation. Here, we have assessed the potential role of peptides on superantigen presentation by (a) analyzing the ability of superantigens to block peptide-specific T cell responses and (b) analyzing the ability of individual peptides to promote superantigen presentation on I-Ab-expressing T2 cells that have a quantitative defect in antigen processing. A series of peptides is described that specifically promote either toxic shock syndrome toxin (TSST) 1 or staphylococcal enterotoxin A (SEA) presentation. Whereas some peptides promoted the presentation of TSST-1 (almost 5,000-fold in the case of one peptide), other peptides promoted the presentation of SEA. These data demonstrate that MHC class II-associated peptides differentially influence the presentation of bacterial superantigens to T cells.

Major histocompatibility complex (MHC) class I ligand motifs have been defined for a number of class I molecules and have been successfully used to identify class I-restricted cytotoxic T-cell epitopes. In contrast, the relative degeneracy of sequence motifs in naturally processed MHC class II ligands has suggested that they may be of more limited use. Here, we use a predicted I-Ab ligand motif to identify antigenic peptides in the Sendai virus Enders strain matrix (M) protein. The entire coding sequence of the M protein was derived, and seven peptide sequences that contained the predicted I-Ab motif were identified. Analysis of I-Ab-restricted M-specific T-cell hybridomas for reactivity to these synthetic peptides identified two distinct epitopes. These data demonstrate that MHC class II motifs can be valuable in predicting T-cell epitopes.

The CTL response to Sendai virus in C57BL/6 mice is directed almost exclusively to a single H-2Kb-restricted epitope derived from the virus nucleoprotein, NP324-332 (FAPGNYPAL). We have previously shown that the repertoire of T cells elicited by this epitope following primary Sendai virus infection is very diverse. The current experiments were undertaken to determine how a diverse array of TCR are able to interact with a single class I epitope. Crystallographic analysis of NP324-332 bound to Kb has shown that the side chains of peptide residues F1, G4, N5, and A8 protrude toward the solvent and are potentially available for recognition by the TCR. Notably, the N5 residue protrudes prominently from the peptide-binding site due to its localization on a bulge in the center of NP324-332. To determine the importance of these residues for T cell recognition, we analyzed the response of a large panel of hybridomas to NP324-332 analogues substituted at these four positions. The data suggested that there is dominant recognition of the central G4 and N5 residues at the center of the peptide. However, individual hybridomas exhibited distinct patterns of fine specificity for residues F1 and A8, in that they were dependent on one, both, or neither of these residues for recognition of NP324-332. These data are consistent with a critical role for the G4 and N5 residues in governing NP324-332/Kb recognition by T cells and may have implications for T cell recognition of class-I restricted epitopes in general.

We sought to determine whether the detectability of phytohemagglutinin-inducible p24 (PHA-p24) in short term cultures of peripheral blood mononuclear cells correlates with an increased risk of vertical transmission among human immunodeficiency virus type 1 (HIV-1)-infected pregnant women and more severe symptomatology among HIV-1-infected infants. The assay for PHA-p24 was performed on specimens obtained from HIV-1-infected women during their pregnancy and from infants during the first 6 months of life. Infants were followed prospectively to determine HIV-1 infection outcome and symptomatology. Among PHA-p24 positive women 9 of 19 (47.4%) gave birth to HIV-1-infected infants compared with 4 of 25 (16.0%) of PHA-p24-negative women (P = 0.02). Among women who tested PHA-p24-positive and had a CD4+ lymphocyte count < 500 cells/mm3, 8 of 15 (53.3%) gave birth to HIV-1-infected infants compared with 4 of 26 (15.4%) not meeting these conditions (P = 0.01). Among HIV-1-infected infants 4 of 5 (80%) of those testing PHA-p24-positive by one month of age developed an opportunistic infection or encephalopathy by 12 months of age, compared with none of the 11 infants testing PHA-p24-negative (P = 0.003). We conclude that PHA-p24 may be a useful in vitro measure for increased risk of vertical transmission among HIV-1-infected pregnant women and increased risk for rapid development of severe disease among HIV-1-infected infants.

We have used Sendai virus infection of C57BL/6 mice as a model with which to study the T cell response to a single MHC class I epitope. Cells taken from the bronchoalveolar lavage or restimulated in vitro from the mediastinal lymph nodes of virus-infected mice were strongly cytotoxic for a single nucleoprotein epitope, NP324-332/Kb. To correlate TCR usage with specificity for the immunodominant epitope, we generated T cell hybridomas from the bronchoalveolar lavage and mediastinal lymph node cells of C57BL/6 mice at the peak of infection. Altogether, 20 hybridomas were identified that specifically secreted IL-2 in response to NP324-332-pulsed L929-Kb cells. TCR usage in this panel of hybridomas was extremely diverse. Over half of the available J beta and V beta elements present in the C57BL/6 strain of mouse were represented in the hybridomas. Similarly, V alpha usage was also diverse and all 12 of the alpha chains sequenced used distinct J alpha elements. The only relatively conserved feature of the TCR in these hybridomas was the presence of an arginine residue in the junctions of 70% of the beta chains. These data demonstrate that a diverse repertoire of TCR is able to recognize a single MHC class I epitope. Moreover, the data demonstrate that mice make use of this potential diversity in the primary response to a natural viral infection.

Sendai virus infection of C57BL/6 mice elicits a strong CD4+ and CD8+ T-cell response in the respiratory tract. To investigate the specificity of the CD4+ T-cell response, a panel of hybridomas was generated from cells recovered from the respiratory tracts of infected mice. Using vaccinia virus recombinants expressing individual Sendai virus proteins, we found that the majority of these hybridomas (34 of 37) were specific for the hemagglutinin-neuraminidase (HN) glycoprotein. The hybridomas were then analyzed for reactivity to a set of overlapping peptides spanning the entire length of the hemagglutinin-neuraminidase glycoprotein. At least five H-2 I-Ab-restricted epitopes were defined in HN. The strong bias toward recognition of class II epitopes derived from a single viral protein contrasts with T-cell recognition of epitopes of several proteins in influenza A virus as found previously by others.

We sought to validate an in vitro system which could predict the minimal effect dose of antiretroviral agents. Mixtures of uninfected CEM cells and CEM cells chronically infected with human immunodeficiency virus (HIV) type 1 MN were exposed to 2',3'-didehydro-3'-deoxythymidine (D4T) in vitro in a hollow-fiber model which simulates the plasma concentration-time profile of D4T in patients. Drug concentration was adjusted to simulate continuous intravenous infusion, or an intravenous bolus administered twice daily. The effect of the dosing regimen was measured with viral infectivity, p24 antigen, and reverse transcriptase or PCR for unintegrated HIV DNA. Dose deescalation studies on a twice-daily dosing schedule predicted a minimum effect dose of 0.5 mg/kg of body weight per day which correlated with the results of a clinical trial. Antiviral effect was demonstrated to be independent of schedule for every 12-h dosing versus continuous infusion. Finally, at or near the minimal effect dose, efficacy appeared to depend on the viral load. The ability of this in vitro pharmacodynamic model to assess the response of HIV-infected cells to different doses and schedules of antiviral agents may be useful in the design of optimal dosing regimens for clinical trials but requires validation with other types of antiretroviral agents.

The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR beta-chain gene (V beta 8.1D beta 2J beta 2.3C beta 2). Analyzing the prevalence of V beta 8.1+ CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3 epsilon showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR beta chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.

Recombinant vaccinia virus expressing the Lassa virus (LV) envelope glycoprotein precursor, V-LSGPC, was used to study the basis of LV-induced cross-protective immunity against the closely related arenavirus lymphocytic choriomeningitis virus (LCMV). C3H/HeJ mice primed with V-LSGPC developed neither circulating antibodies nor CD8+ cytotoxic T cells specific for LCMV, yet they resisted a normally lethal LCMV challenge. Spleen cells from such mice gave a proliferative response to LCMV in vitro that was inhibitable by anti-CD4 antibody. Synthetic peptides corresponding to predicted T-cell sites common to the envelope glycoprotein precursor (GP-C) of LV and that of LCMV were used to map the specificity of the proliferative response to an epitope located between amino acids 403 and 417 of LV GP-C. Several CD4+ T-cell clones specific for the 403-417 peptide were isolated and found to produce gamma interferon in response to both the peptide and LCMV. One of these clones, C9, was selected for further study. C9 lysed I-AK-bearing target cells, and when adoptively transferred to C3H/HeJ mice, it was capable of mediating both a peptide-specific delayed hypersensitivity reaction and resistance to lethal LCMV challenge. These collective findings demonstrate, for the first time, that CD4+ T cells can play a major role in arenavirus-specific cross-protective immunity.

The effects of two adenosine diphosphoribose transferase (ADPRT) enzyme inhibitory ligands, 6-amino-1,2-benzopyrone and its 5-iodo-derivative, were determined in AA-2 and MT-2 cell cultures on the replication of HIV-1 IIIb, assayed by an immunochemical test for the HIV protein p24, and syncytium formation, characteristic of HIV-infected cells. Intracellular concentrations of both drugs were sufficient to inhibit poly(ADP-ribose) polymerase activity within the intact cell. Both drugs inhibited HIV replication parallel to their inhibitory potency on ADPRT, but distinct differences were ascertained between the two cell lines. In AA-2 cells both p24 and syncytium formation were depressed simultaneously, whereas in MT-2 cells only syncytium formation was inhibited by the drugs, and the p24 production, which remained unchanged during viral growth, was unaffected. Both drugs only moderately depressed the growth rate of the AA-2 and MT-2 cells and there was no detectable cellular toxicity. Results suggest the feasibility of the development of a new line of ADPRT ligand anti-HIV drugs that fundamentally differ in their mode of action from currently used chemotherapeutics.

In previous studies, the murine SaI (A/J derived, KkDd) sarcoma was transfected with the allogeneic MHC class I H-2Kb gene, and expressed high levels of H-2Kb antigen. Contrary to expectations, the tumor cells expressing the alloantigen (SKB3.1M tumor cells) were not rejected by autologous A/J mice. Because these results contradict the laws of transplantation immunology, the present studies were undertaken to examine the immunogenicity of SKB3.1M and SaI cells in allogeneic hosts. Similar to SKB3.1M, SaI cells are lethal in some allogeneic strains, despite tumor-host MHC class I incompatibilities. Tumor challenges of SKB3.1M and SaI cells, however induce MHC class I-specific antibodies and CTL in both tumor-resistant and -susceptible hosts. Although the tumors induce specific CTL, tumor cells are not lysed in vitro by these CTL, suggesting that the tumor cells are resistant to CTL-mediated lysis. Since growth of these tumors does not follow the classical rules of allograft transplantation, and because the tumor is not susceptible to CTL-mediated lysis, we have used Winn assays to identify the effector lymphocyte(s) responsible for SaI rejection. Depletion studies demonstrate that the effector cell is a CD4-CD8+ T lymphocyte. Collectively these studies suggest that the host's response to MHC class I alloantigens of SKB3.1M and SaI cells does not determine tumor rejection, and that effector cells other than classically defined CTL, but with the CD4-CD8+ phenotype, can mediate tumor-specific immunity.

Mouse Sal sarcoma cells are lethal in the autologous A/J (KkDd) host. In order to improve the immune response to the Sal tumor, Sal cells have been transfected with syngeneic MHC-class-II or allogeneic MHC-class-I genes. MHC-class-II transfectants are uniformly rejected by the autologous host and immunization with them protects against subsequent Sal challenge. The improved immunity is probably the result of enhanced generation of tumor-specific Th cells. We hypothesize that class-II tumor cells trigger an improved Th-cell response because they directly present Sal tumor antigens in the context of class-II molecules to Th cells, by-passing professional APC. Studies by others have demonstrated that antigen presentation requires an intracellular signal transmitted by the cytoplasmic domain of the APC class-II molecule. Sal cells expressing class-II antigens with truncated cytoplasmic domains are as malignant as wild-type Sal cells. These experiments therefore support the role of tumor-cell class-II molecules as antigen presentation elements, and demonstrate the requirement for intact class-II molecules for tumor protection. Sal cells have also been transfected with allogeneic MHC-class-I genes. Although Kb-transfected cells are not rejected by A/J mice, Db-transfected Sal cells and Kb- plus Db-transfected cells are rejected. The Db transfectants effectively immunize A/J mice against subsequent Sal challenge. These experiments demonstrate that expression of certain allogeneic MHC-class-I genes can lead to tumor-specific immunity, and that such transfectants can protect against challenges of wild-type tumor cells. Transfection of tumor cells with syngeneic MHC-class-II or allogeneic MHC-class-I genes may therefore be a potential strategy for improving tumor-specific immunity in the autologous host.

Inhibitory factors to human immunodeficiency virus type 1 (HIV-1) in saliva may be responsible for the infrequent isolation of virus from saliva and also may account for the marked infrequency of salivary and/or oral transmission of HIV-1. Incubation of HIV-1 with human saliva followed by addition of the mixture to susceptible cells leads to partial or complete suppression of viral replication in vitro. We investigated the inhibitory effects of whole saliva and specific glandular salivas on HIV-1 infectivity as measured by viral-induced cytopathic effects in susceptible cells. Whole saliva contained marked inhibitory activity to HIV-1, strain HTLV-IIIB, and to virus infected cells. Submandibular saliva contained inhibitory activity, but of lesser quantity. Parotid saliva demonstrated no HIV-inhibitory activity. Whole saliva also appeared to contain filterable components that were inhibitory to lymphocyte growth. Passage through a .45 micron pore-size filter eliminated the viral inhibitory activity of submandibular saliva and some of the activity in whole saliva. All salivas except parotid incubated with HIV-1 followed by filtration were inhibitory suggesting that complexing of virus with high molecular weight, submandibular mucins may play a role in viral inhibition.

Rejection of the MHC class I negative 402AX teratocarcinoma is accompanied by induction of tumor cell-encoded H-2K and H-2D antigens by the genetically resistant host. To determine whether MHC antigen expression is required for 402AX rejection, we have prepared H-2Db-transfected 402AX cells (402AX/Db). Transfectants express high levels of H-2Db, most of which is not associated with beta 2-microglobulin. MHC syngeneic and allogeneic mice susceptible to 402AX are resistant to 402AX/Db, suggesting that MHC class I antigen expression is required for tumor rejection. Autologous 129 hosts, however, are susceptible to 402AX/Db. 402AX cells transfected with the H-2Kb gene (402AX/Kb) are also lethal in the autologous 129/J host, but rejected by MHC syngeneic and allogeneic mice. Non-129 strain 402AX-susceptible mice pre-immunized with 402AX/Db or simultaneously challenged with 402AX/Db plus 402AX are immune to 402AX. Mice immunized with 402AX/Db produce MHC class I induction factor. 402AX/Db and 402AX cells are lysed equally by natural killer cells, indicating that in 402AX cells the expression of class I antigens is unrelated to NK susceptibility. These studies confirm the requirement for class I expression in 402AX immunity, but demonstrate that in the autologous host immunity requires additional factors beyond class I antigen expression.

Lymphocytic choriomeningitis (LCM) virus of the mice provides perhaps the most extensively studied model of viral-induced immunopathology. Intraocular inoculation of this virus in the adult mouse produces a severe and progressive uveitis with involvement also of the cornea and retina. Intracerebral infection of the newborn rat with LCM virus produces a severe retinopathy, with only minimal involvement of the uveal tract. We review in this paper recent studies on these two animal models of LCM virus disease which demonstrate: (1) the histopathology of the ocular lesion; (2) that immunosuppression after infection prevents development of the disease; (3) that passive transfer of sensitized lymphoid cells to the immunosuppressed infected animal can then trigger ocular disease, and (4) that T lymphocytes, and not circulating antibody, play the major role in the pathogenesis of this disease process.

Cloned lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T lymphocyte (CTL) lines were prepared from spleens of 129/J (H-2b) mice immunized 7-9 months earlier with LCMV (UBC strain), or of C57BL/10J (H-2b) mice immunized 4 to 5 weeks earlier with LCMV (Armstrong strain). One uncloned and 3 cloned cytotoxic T cell lines were assessed for their respective abilities to produce, or protect against, fatal disease upon transfer to appropriate recipients or to induce specific footpad-swelling reaction. The effects of all lines were essentially identical. In recipient mice acutely infected with LCMV and immunosuppressed either by irradiation (750-990 rds) or treatment with cyclophosphamide, cloned T cells administered intracerebrally (i.c.) caused a convulsive disease and death within 1-4 days. No disease was produced when the same CTL were transferred to uninfected recipients or when they had been frozen and thawed prior to transfer to infected recipients. When admixed with 500 plaque-forming units of LCMV and transferred i.c. to immunocompetent H-2b mice, the T cell clones prevented overt disease. Allogeneic (H-2k) recipients of this same admixture all developed typical LCM disease as did H-2b recipients of the admixture after T cells had been frozen and thawed. Inoculation of cloned CTL into preinfected footpads induced a specific footpad-swelling reaction, which reached maximum levels after about 36 h. Irradiated and infected recipients of cloned LCMV-specific T cells showed the footpad-swelling reaction only when they had been reconstituted with bone marrow cells. In contrast, cloned T cells induced LCM disease in i.c. infected and irradiated mice independent of bone marrow reconstitution. These findings indicate that both fatal LCMV-induced neurologic disease and protection against it are mediated directly by virus-specific CTL.

Sindbis virus variants evidencing a complex and bidirectional tendency toward spontaneous antigenic change were isolated and characterized. Variants were selected on the basis of their escape from neutralization by individual monoclonal antibodies to either of the two envelope glycoproteins, E2 and E1. Multisite variants, including one altered in three neutralization sites, were obtained by selecting mutants consecutively in the presence of different neutralizing monoclonal antibodies. Two phenotypic revertants, each of which reacquired prototype antigenicity, were back-selected on the basis of their reactivity with a neutralizing monoclonal antibody. An incidental oligonucleotide marker distinguished these and the variant from which they arose from parental Sindbis virus and other mutants, thereby confirming that the revertants were true progeny of the antigenic variant. Prototype Sindbis virus and variants derived from it were compared on the basis of their reactivities with each of a panel of monoclonal antibodies; patterns revealed a minimum of five independently mutable Sindbis virus neutralization epitopes, segregating as three antigenic sites (two E2 and one E1).

The nature of the interaction between vaccinia virus (VAC) and fibroblastic cells that renders the latter capable of being recognized by virus-specific, H-2 identical murine T lymphocytes has been studied. L cells exposed for 10 min to VAC rendered noninfectious by exposure to ultraviolet light became susceptible targets for cytotoxic T lymphocytes (CTL) without the synthesis of new viral proteins. Susceptibility was retained even if cellular protein synthesis was irreversibly inhibited with pactamycin before virus exposure. Immobilization of cell-surface membranes by glutaraldehyde fixation before (but not after) exposure to virus severely impaired the formation of the "virus + self" complex that in vitro stimulated secondary CTL responses by H-2 identical virus-primed memory cells even though virus attachment to fixed cells were unaffected. This stimulatory complex, once formed, was maintained in membrane fragments prepared from cells previously exposed to VAC. These findings indicate that VAC-specific CTL or their immediate precursors can recognize only those viral envelope antigens that become membrane integrated and that this event requires neither host cell-specific nor virus-specific protein synthesis.

Hyperplasia of endocrine cells in the lung of the adult rat exposed to asbestos has only been characterised so far by electron microscopy as there is a lack of reliable staining techniques for their demonstration at light microscopical level. Neuron specific enolase (NSE), an isoenzyme of the glycolytic enzyme enolase has recently been shown to be present in lung endocrine cells. In this study we reveal a marked endocrine cell hyperplasia at light microscopical level in the lungs of adult rats exposed to asbestos using antibodies to NSE. Very large groups of NSE-immunoreactive cells (20-80) were only observed in the lungs of rats exposed to asbestos for 12 months. In addition smaller groups of cells (2-10) known to be present normally and to decrease with age, were rarely noted in the controls but were frequently detected in the treated rats. Immunoreactive NSE is therefore a very good marker for endocrine cell hyperplasia and thus of early neoplastic changes.

There exists in the mouse a family of I-region-controlled (Ia) antigens which carry carbohydrate-defined determinants. These antigens appear in serum as glycolipids and seem to be actively secreted by antigen-activated T-cells. This paper describes the ability of selected viral, bacterial, and protozoal infections of mice to markedly alter the serum levels of these Ia antigens. All the infectious agents examined induced substantial augmentation or suppression of serum Ia concentrations or both. Lymphocytic choriomeningitis (LCM) virus first enhanced and then suppressed serum Ia levels during the course of acute infection. Enhancement occurred during the time of ongoing virus replication and splenic lymphoproliferation while suppression coincided with the peak of the cytotoxic T-cell response and virus clearance. Listeria monocytogenes infection induced a substantial reduction in Ia levels at a time just after marked depletion of T-cells in the spleen. In contrast, Brucella abortus caused a significant increase in Ia levels 7 days postinfection, which correlates with the appearance of peak numbers of bacteria in tissues. Finally, Plasmodium yoelii, a nonlethal malarial parasite which stimulates prolonged T-cell proliferation, augmented serum Ia levels, whereas P. berghei, a lethal parasite which tends to inhibit. T-cell division, suppressed Ia secretion. Possible interpretations of these different results are presented.