Sources of variation in rates of in vitro ruminal protein degradation.

Rates and extents of ruminal protein degradation for casein, solvent soybean meal (SSBM), expeller soybean meal (ESBM), and alfalfa hay were estimated from net appearance of NH3 and total amino acids in in vitro media containing 1 mM hydrazine and 30 mg/L of chloramphenicol. Protein was added at 0.13 mg of N/mL of medium, and incubations were conducted for 4 to 6 h, usually with hourly sampling. Inocula were obtained from ruminally cannulated donor cows fed diets of grass silage or alfalfa and corn silages plus concentrates. Preincubation or dialysis of inocula was used to suppress background NH3 and total amino acids; however, preincubation yielded more rapid degradation rates for casein and SSBM and was used in subsequent incubations. Preincubation with added vitamins, VFA, hemin, or N did not alter protein degradation. Protein degradation rates estimated for SSBM, ESBM, and alfalfa were not different when computed from total N release or N release in NH3 plus total amino acids, regardless of whether amino acids were quantified using ninhydrin colorimetry or o-phthalaldehyde fluorescence. Accounting for the release of peptide-N also did not affect estimated degradation. However, casein degradation rates were more rapid when using total N release or accounting for peptide-N, indicating significant accumulation of small peptides during its breakdown. Rates also were more rapid with inocula from lactating cows versus nonlactating cows with lower feed intake. Protein degradation rates were different due to time after feeding: casein rate was more rapid, but SSBM and ESBM rates were slower with inocula obtained after feeding. Several characteristics of ruminal inoculum that influenced breakdown of the rapidly degraded protein casein did not appear to have direct effects on degradation of protein in soybean meal.

IL-10 mediates susceptibility to Leishmania donovani infection.

Human visceral leishmaniasis (VL) results in a severe and potentially fatal systemic disease, accompanied by cellular immune depression. The production of IL-10 correlates with ongoing disease and it has been suggested that the cellular immune depression that accompanies active disease may be due to a predominance of IL-10 production rather than a lack of IFN-gamma production, which is essential for optimal macrophage activation and parasite elimination. To examine the role of IL-10 in resistance during L. donovani infection (a causative agent of VL), the course of infection was examined in mice lacking the gene for IL-10. BALB/c IL-10-/-, as well as C57BL/6 IL-10-/- mice, were highly resistant to L. donovani infection, as evidenced by liver parasite burdens which were tenfold lower than those in control mice after 14 days of infection. Enhanced resistance was accompanied by increased production of IFN-gamma and nitric oxide in BALB/c IL-10-/- mice. Susceptibility to infection in BALB/c IL-10-/- mice was enhanced following in vivo treatment with a neutralizing antibody to IFN-gamma or IL-12. Together these studies demonstrate for the first time that IL-10 is a critical component of the immune response that inhibits resistance to L. donovani.

Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice.

Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.

A prospective observational study of 5-, 7-, and 10-day antibiotic treatment for acute otitis media.

OBJECTIVE: To compare 5-, 7- and 10-day duration of antibiotic therapy for acute otitis media (AOM) in children. STUDY DESIGN AND SETTING: Prospective nonrandomized 1-year evaluation of 3 treatment durations for AOM in a private pediatric setting. Outcomes assessed at 14 +/- 4 days after start of therapy with clinical response categorized as cure, improvement, or failure. RESULTS: A total of 2172 children were studied; 46.4% were < or =2-years-old. Antibiotics used were amoxicillin (61.9% of patients), trimethoprim/sulfamethoxazole (11.7%), cephalosporins (14.2%), amoxicillin/clavulanate (5.2%), and macrolides/azalides (4.8%). No overall difference in outcome was observed comparing the 5-day (n = 707), 7-day (n = 423), or 10-day (n = 1042) treatments, including children < or =2-years-old. However, in the subset who had an episode of AOM in the preceding month, outcome differed; 5-day treatment was followed by more frequent failure than 10-day treatment (P < 0.001). In logistic regression analysis, variables identified as contributing to a cure were: >2-years-old (P < 0.0001), no AOM in the preceding month (P = 0.07), or preceding 12 months (P = 0.03). CONCLUSIONS: Our study supports the transition from 10 to 5 days for standard AOM antibiotic treatment duration in most patients. A 10-day regimen may be superior in children who have experienced an episode of AOM within the preceding month, a known risk factor for resistant bacterial infection in the otitis-prone patient.

Correlation of preoperative computed tomography and postoperative ocular motility in orbital blowout fractures.

PURPOSE: To determine a relationship between preoperative soft tissue disruption and postoperative ocular motility in orbital blowout fractures. METHODS: This retrospective cohort study reviewed 30 patients who met all criteria: retrievable coronal computed tomography (CT) scans; internal fractures of the orbital floor, with or without medial wall extension; preoperative diplopia; repair by a single surgeon; complete release of entrapped tissues; and postoperative binocular visual fields (BVFs). Motility outcomes were quantified by one group of the authors, who measured the vertical fusion within BVFs. Other authors analyzed CT scans, designating each fracture as either A or B, based on lesser or greater soft tissue distortion relative to the configuration of bone fragments. The interval between trauma and surgery was also determined. RESULTS: Among the 15 patients with a postoperative motility outcome poorer than the median (86 degrees or less), four (27%) had A fractures; 11 (73%) had B fractures. Among the 15 patients with an outcome better than the median (88 degrees or more), 10 (67%) had A fractures; five (33%) had B fractures. Differences were more defined away from the median. Among five patients with B fractures and better than the median result, three (60%) had surgical repair during the first week after injury. Among the 11 patients with B fractures and less than the median result, one (9%) had repair during the first week. CONCLUSIONS: Postoperative motility is influenced by soft tissue-bone fragment relationships. Whether the outcome can be altered by earlier surgery in selected cases will be determined by prospective studies.

Outcomes after judicious antibiotic use for respiratory tract infections seen in a private pediatric practice.

BACKGROUND: Most respiratory tract infections (RTIs) in children have a viral cause, they resolve on their own, and antibiotics need not be prescribed. OBJECTIVE: We sought to provide evidence that judicious antibiotic use can be accomplished in private pediatric practice without observing an increase in return office visits or in the rate of bacterial infections that may follow. STUDY DESIGN: This was a prospective 12-month study from July 1, 1996 through June 30, 1997. On the same 1 day each week, a representative convenience sample of acute respiratory tract illness patients was enrolled, and laboratory studies performed as appropriate, including viral cultures on all. Children were then followed for 30 days to ascertain the outcomes of not prescribing antibiotics except when specific bacterial infections were present at the initial visit. RESULTS: Three hundred eighty-three children were enrolled; 293 (77%) did not receive antibiotics at the enrollment visit. Ninety children (23%) received antibiotics based on a diagnosis of acute otitis media (n = 53), acute streptococcal tonsillopharyngitis (n = 18), or other presumed or documented bacterial infections (n = 19). An unscheduled return visit related to the initial visit occurred for 86 (29%) of the 293 children not receiving antibiotics initially and in 40 (44%) of 90 children receiving antibiotics initially. Eighty-seven children (23%) had positive viral culture results. The most frequently isolated viruses were adenovirus, enterovirus, parainfluenzae virus, and influenza virus. CONCLUSION: Children with RTIs without a concomitant presumed or proven bacterial infection do not require antibiotics. In this busy office practice, >75% of the children presenting with an RTI did not have a presumed or proven bacterial infection. These children did not have a higher rate of return office visits or an increase in bacterial infections. This reinforces the judicious use of antibiotics in managing children with RTIs.outcomes, antibiotic, respiratory infections.

Localization of foot-and-mouth disease virus RNA by in situ hybridization within bovine tissues.

Foot-and-mouth disease is a highly contagious disease of cloven hooved animals. In cattle, both acute and long-term persistent infections occur. Foot-and-mouth disease virus (FMDV), a picornavirus, has been shown, using virus isolation procedures, to replicate in the pharynx and soft palate of cattle. In this study, in situ hybridization has been used to detect FMDV RNA within the cells of tissues removed from infected bovines. A digoxigenin-labelled anti-sense RNA probe was prepared corresponding to a region of the FMDV genome encoding part of the RNA-dependent RNA polymerase (3D). The efficacy and specificity of this probe for in situ hybridisation was determined using virus-infected cells in tissue culture. Strong cytoplasmic staining was only detected in FMDV-infected cells. Various tissue samples were collected from FMDV-infected cattle between 5 and 17 days post-infection. Viral RNA was detected by in situ hybridisation within cells of the soft palate, tonsil and pharynx up to 17 days post-infection. This technique is useful for the study of FMDV localization in cattle both during and after the acute clinical phase of disease and may assist in identifying specific sites of virus persistence.

Incidence of streptococcal carriers in private pediatric practice.

OBJECTIVE: To determine the incidence of group A beta-hemolytic streptococcus (GABHS) carriers in children who are well, in children seen with presumed and documented viral illnesses with sore throat, and in children after treatment of acute GABHS tonsillopharyngitis with 10 days of oral penicillin V potassium, oral cephalosporins, or macrolides. METHODS: Prospective collection of clinical and microbiologic data from October 1996 to June 1997 in a private pediatric practice were obtained from children who were asymptomatic and well, from children with both presumed (and documented) viral sore throats, and from children who had completed a full antibiotic treatment course for acute GABHS throat infections. RESULTS: The incidence of GABHS carriers was 2.5% among well children (n = 227), 4.4% among children with upper respiratory tract infections including sore throat of presumed viral etiology (n= 296), and 6.9% among children with upper respiratory tract infections including sore throat from whom viruses were isolated (n = 87). Following 10 days' treatment of acute GABHS tonsillopharyngitis, 81 (11.3%) of 718 children treated with penicillin, 22 (4.3%) of 508 children treated with an oral cephalosporin, and 10 (7.1%) of 140 children treated with a macrolide were GABHS carriers (P<.001). CONCLUSIONS: A small percentage of children seen in private pediatric practices who are well or who have apparent viral upper respiratory tract infections with sore throat are GABHS carriers. Penicillin treatment of acute GABHS tonsillopharyngitis results in a higher GABHS carriage rate than occurs following treatment with cephalosporins and macrolides.

Blockade of CTLA-4 enhances host resistance to the intracellular pathogen, Leishmania donovani.

CTLA-4 has recently been shown to act as a negative regulator of T cell activation. Here we provide evidence that blockade of CTLA-4 can result in enhanced host resistance to an intracellular pathogen. The administration of anti-CTLA-4 mAb 4F10 to BALB/c mice, 1 day following infection with Leishmania donovani, enhanced the frequency of IFN-gamma and IL-4 producing cells in both spleen and liver, and dramatically accelerated the development of a hepatic granulomatous response. The expression of mRNA for the CXC chemokine gammaIP-10 was also elevated above that seen in control Ab treated mice, and was directly correlated with the frequency of IFN-gamma producing cells. In contrast, macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1) mRNA levels were unaffected by anti-CTLA-4 treatment, suggesting that CTLA-4 blockade may exert selective effects on chemokine expression. These changes in tissue response and cytokine/chemokine production were accompanied by a 50 to 75% reduction of parasite load in the spleen and liver of anti-CTLA-4-treated animals compared to controls. Furthermore, administration of anti-CTLA-4 mAb 15 days after L. donovani infection, when parasite burden is increasing in both organs, also resulted in enhanced resistance. Thus, these studies indicate a potent immunomodulatory and potentially therapeutic role for interventions targeted at CTLA-4.

Neutralization of IL-12 demonstrates the existence of discrete organ-specific phases in the control of Leishmania donovani.

IL-12 plays a key role in stimulating both innate and antigen-specific immune responses against a number of intracellular pathogens. A neutralizing anti-IL-12 monoclonal antibody (mAb) was used to define and compare the role of endogenous IL-12 in the liver and spleen of mice infected with Leishmania donovani. IL-12 neutralization both early and late in infection caused delayed resolution of parasite load, a transient decrease in IFN-gamma, IL-4, TNF-alpha and inducible nitric oxide synthase (NOS-2) production, and suppressed tissue granuloma formation in the liver of genetically susceptible BALB/c mice. In contrast to the liver of BALB/c mice, neutralization of IL-12 had no effect on parasite burden in the spleen over the first 28 days of infection. However, IL-12 appeared to be critical for the development of mechanisms which subsequently contain the growth of persistent parasites in this organ in that neutralization of IL-12 dramatically enhanced parasite growth after day 28 of infection. Following IL-12 neutralization, the later unchecked growth of parasites in the spleen was coincident with an extensive breakdown of the tissue microarchitecture. Immunohistochemical studies revealed that IL-12 was largely produced by uninfected cells in L. donovani-infected BALB/c mice. In contrast, the course of infection in the liver and spleen of genetically resistant CBA/n mice was unaffected by the administration of anti-IL-12 mAb. These results suggest that the liver and spleen in susceptible BALB/c mice have different temporal requirements for IL-12 in controlling L. donovani infection, whereas IL-12 plays little role in either organ in resistant CBA/n mice. In addition, IL-12 appears to be involved in the generation of both Th1 and Th2 responses during L. donovani infection in BALB/c mice.

Orbital blow-out fractures: correlation of preoperative computed tomography and postoperative ocular motility.

BACKGROUND/PURPOSE: Although the management of orbital blow-out fractures was controversial for many years, refined imaging with computed tomography (CT) helped to narrow the poles of the debate. Many orbital surgeons currently recommend repair if fracture size portends late enophthalmos, or if diplopia has not substantially resolved within 2 weeks of the injury. While volumetric considerations have been generally well-served by this approach, ocular motility outcomes have been less than ideal. In one series, almost 50% of patients had residual diplopia 6 months after surgery. A fine network of fibrous septa that functionally unites the periosteum of the orbital floor, the inferior fibrofatty tissues, and the sheaths of the inferior rectus and oblique muscles was demonstrated by Koornneef. Entrapment between bone fragments of any of the components of this anatomic unit can limit ocular motility. Based on the pathogenesis of blow-out fractures, in which the fibrofatty-muscular complex is driven to varying degrees between bone fragments, some measure of soft tissue damage might be anticipated. Subsequent intrinsic fibrosis and contraction can tether globe movement, despite complete reduction of herniated orbital tissue from the fracture site. We postulated that the extent of this soft tissue damage might be estimated from preoperative imaging studies. METHODS: Study criteria included: retrievable coronal CT scans; fractures of the orbital floor without rim involvement, with or without extension into the medial wall; preoperative diplopia; surgical repair by a single surgeon; complete release of entrapped tissues; and postoperative ocular motility outcomes documented with binocular visual fields (BVFs). Thirty patients met all criteria. The CT scans and BVFs were assessed by different examiners among the authors. Fractures were classified into 3 general categories and 2 subtypes to reflect the severity of soft tissue damage within each category. "Trap-door" injuries, in which bone fragments appeared to have almost perfectly realigned, were classified as type I fractures. In the I-A subtype, no orbital tissue was visible on the sinus side of the fracture line. In the I-B subtype, soft tissue with the radiodensity of orbital fat was visible within the maxillary sinus. In type II fractures, bone fragments were distracted and soft tissue was displaced between them. In the II-A subtype, soft tissue displacement was less than, or proportional to, bone fragment distraction. In the II-B subtype, soft tissue displacement was greater than bone fragment distraction. In type III fractures, displaced bone fragments surrounded displaced soft tissue in all areas. In the III-A subtype, soft tissue and bone were moderately displaced. In the III-B subtype, both were markedly displaced. Motility outcomes were quantified by measuring the vertical excursion in BVFs. The interval between trauma and surgical repair was also determined. RESULTS: Among the 15 patients with a motility outcome in BVFs which was poorer than the median (86 degrees or less of single binocular vertical excursion), 4 patients (27%) had type A fractures; 11 patients (73%) had type B fractures. Among the 15 patients with a better outcome than the median (88 degrees or more), 10 patients (67%) had type A fractures; 5 patients (33%) had type B fractures. These differences became more defined as analysis moved away from the median. Among 5 patients with type B fractures and better than the median result in BVFs, 3 patients (60%) had surgical repair during the first week after injury. Among the 11 patients with type B fractures and less than the median result, 1 patient (9%) had repair during the first week. CONCLUSIONS: When the CT-depicted relationship between bone fragments and soft tissues is considered, a wide spectrum of injuries is subsumed under the rubric of blow-out fractures. In general, greater degrees of soft tissue incarceration or displacement, with presumably greater intrinsic damage and subsequent fibrosis, appear to result in poorer motility outcomes. Although this retrospective study does not conclusively prove its benefit, an urgent surgical approach to selected injuries should be considered.

B7-2 blockade enhances T cell responses to Leishmania donovani.

Infection with Leishmania donovani has been reported to induce a dominant Th1-type response in all strains of mice examined, providing a model for examining the regulation of Th1 responses in the relative absence of Th2 cross-regulation. Here we demonstrate that blockade of the costimulatory molecule B7-2, but not B7-1, has significant effects on disease progression, measured as day 28 parasite burden in the liver. The effects of B7-2 blockade were associated with increased IFN-gamma production, as determined 1) following restimulation with specific Ag, 2) by enumeration of IFN-gamma-secreting cells using ELISPOT assays, and 3) by analysis of IFN-gamma mRNA by reverse transcription-PCR. Surprisingly, IL-4-producing cells were also readily detected in infected mice by ELISPOT analysis. The frequency of these IL-4-producing cells and of IL-4 mRNA levels was also enhanced in the liver of infected mice treated with anti-B7-2 mAb. Administration of anti-B7-2 from the day of infection or delaying its administration until day 3 after infection had similar effects. Parasite-specific IgG1 and IgG2a responses were either unaffected or marginally increased following anti-B7-2 administration, contrary to the inhibitory effect of this treatment on responses to the T-dependent Ag DNP-BSA. These data support a model of T cell activation whereby B7-2/CD28 interactions play a relatively redundant role in initial T cell activation, but in which interference with later B7-2/CTLA4 interaction potentiates established cytokine responses.

Localization of foot and mouth disease virus RNA in tissue culture infected cells via in situ polymerase chain reaction.

Foot and mouth disease virus RNA was visualized in infected primary tissue culture cells by in situ PCR incorporating digoxigenin-labeled dUTP. The viral RNA polymerase gene was used as a target for amplification. Infected cells revealed cytoplasmic staining, predominantly perinuclear. The intensity of staining was in proportion to the degree of cytopathology observed and similar to the results obtained using immunoperoxidase staining. The in situ PCR technique for FMDV detection could be applied to formalin-fixed samples and be useful for the study of persistent infections.

Echographic pseudoextension of uveal melanomas.

BACKGROUND: Standardized echography is routinely utilized to assess uveal melanomas. Echographic pseudoextension is defined as normal structures mimicking intrascleral or extrascleral extension of tumor on echography. METHODS: The records of 151 consecutive uveal melanoma patients evaluated with standardized echography over a 6-year period (1986-1991) were reviewed to identify those in which pseudoextension or true extension was diagnosed. RESULTS: Fourteen (9%) cases of pseudoextension were noted, with causes including juxtapapillary tumor location (seven cases), extraocular muscle insertion (five cases), vortex ampullae (one case), and post-brachytherapy changes (one case). Clinical, echographic, and/or histopathologic follow-up confirmed absence of true extension. Six (4%) cases of true extrascleral extension were identified and confirmed histopathologically. CONCLUSION: Differentiating extraocular tumor extension from pseudoextension is critical, and use of standardized A-scan and contact B-scan echography is integral in this assessment.

A modified en bloc excision technique during vitrectomy for diabetic traction retinal detachment. Results and complications.

PURPOSE: The visual results and complications were determined for a modified en bloc excision technique for management of diabetic traction macular detachment. PATIENTS AND METHODS: A consecutive series of 30 eyes with this condition underwent pars plana vitrectomy, during which all posterior hyaloid except for portions essential for membrane dissection were excised. Bimanual dissection techniques then allowed excision of fibrovascular membranes "en bloc" with the retained hyaloid. RESULTS: After 6 to 44 months of follow-up, visual acuity of 5/200 or better was obtained in 23 (77%) of 30 eyes, with complete reattachment in 29 (97%) of 30 eyes. Iatrogenic retinal breaks occurred in 6 (20%) of 30 eyes. Two eyes required repeat vitrectomy. CONCLUSIONS: The modified en bloc excision technique allowed similar visual outcome, a higher reattachment rate, and less postoperative morbidity than previously reported surgical approaches to diabetic traction macular detachment.

Orbital myositis as a paraneoplastic syndrome.

We describe a patient with bilateral orbital myositis, multiple cranial neuropathies, a sensory polyneuropathy, serum and cerebrospinal fluid paraproteins, and high-grade non-Hodgkin's lymphoma. Neurologic symptoms began more than 1 year before diagnosis of the lymphoma. Results of extraocular muscle biopsy showed extensive destruction of myofibers and granulomatous features, with no evidence of direct tumor involvement. The cranial neuropathies and orbital myositis improved with immunosuppressive therapy, while the patient's tumor progressed. We believe the orbital myositis and the multiple neurologic abnormalities were paraneoplastic effects of the lymphoma. To our knowledge, this is the first case of orbital myositis identified as a paraneoplastic syndrome.

Analysis of sites of foot and mouth disease virus persistence in carrier cattle via the polymerase chain reaction.

This study was undertaken in order to explore possible sites of foot-and-mouth disease virus (FMDV) persistence during the carrier state. Tissue samples taken from experimentally infected animals at different times post-infection (p.i.) were examined by conventional viral isolation and the polymerase chain reaction (PCR) technique. The analysis of samples from several organs taken from 17 bovines between 3 and 270 days p.i. allowed the following conclusions: 1) Virus present in oesophageal-pharyngeal fluids (OPF) during the carrier state originates in the pharynx as shown by the detection of antisense FMDV RNA by PCR, 2) PCR is more sensitive than standard virus isolation techniques and may be used for the rapid detection of FMDV in specimens obtained during the acute stage of FMD and for identification of persistently infected cattle.

Ultrastructural changes in rat mammotropes following incubation with dopamine.

Cultured mammotropes incubated with dopamine for one hour exhibited changes in ultrastructure indicative of actively depressed biosynthetic and secretory activity. Peripheral relocation of rough endoplasmic reticulum appeared to create a barrier to secretory granule release by exocytosis. A decrease in the numbers of secretory granules indicated a decrease in prolactin production and enhanced lysosomal activity.