TCR engagement in the absence of cell cycle progression leads to T cell anergy independent of p27(Kip1).

We have proposed a model in which the prevention of anergy by costimulation is the result of IL-2-induced G1 to S phase cell cycle progression. Here we demonstrate that the reversal of anergy by exogenous IL-2 also occurs during this window of the cell cycle. Recently, it has been proposed that the cell cycle inhibitor p27(Kip1) is an anergic factor. In contrast, our data demonstrate that during the induction, maintenance and rechallenge phases of anergy, p27(Kip1) levels do not correlate with the anergic phenotype. Although p27(Kip1) levels were down-regulated by IL-2 during the G1 to S phase transition, the amount of IL-2 required to produce this effect was far lower than that required to prevent the induction of anergy. Furthermore, T cell lines from p27(Kip1) knockout mice were anergized as well as T cells from mice that were heterozygous for p27(Kip1). Interestingly, the forced overexpression of p27(Kip1) was able to decrease IL-2 promoter-induced transcription, suggesting that the cell cycle machinery may be involved in T cell activation; however, physiological levels of p27(Kip1) did not prevent IL-2 transcription. Overall, our data serve to disassociate the ability of IL-2 to down-regulate p27(Kip1) and its ability to prevent or reverse anergy.

N-terminal processing is essential for release of epithin, a mouse type II membrane serine protease.

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.

Adaptive tolerance of CD4+ T cells in vivo: multiple thresholds in response to a constant level of antigen presentation.

The in vivo T cell response to persistent Ag contains a hyporesponsive phase following an initial expansion and subsequent partial deletion of the responding cells. The mechanism(s) responsible for this tolerance process is poorly understood. In this study, we describe a new paired transgenic model (TCR and Ag), which within 7-14 days produces 20-40 million hyporesponsive T cells. This state is characterized by an 85-95% reduction in all cytokine production, an impairment of re-expression of CD25 and CD69, and a desensitization of the proliferative response to Ag. TCR levels were normal, and in vivo mixing experiments showed no evidence for active suppression. The hyporesponsiveness partially dissipated without proliferation when the cells were transferred into a non-Ag-bearing host. If the second host expressed Ag, the T cells initially regained responsiveness, but then slowly entered an even deeper state of tolerance characterized by an additional 7- to 10-fold lowering of cytokine production and a greater desensitization of proliferation. Surprisingly, this readaptation took place with the same level of Ag presentation, suggesting that other parameters can influence the tolerance threshold. Both the readjustment in sensitivity and the reversal without Ag convincingly demonstrate for the first time a truly adaptive tolerance process in CD4+ T cells in vivo.

Cefprozil versus high-dose amoxicillin/clavulanate in children with acute otitis media.

BACKGROUND: The recommendation of the Drug-Resistant Streptococcus pneumoniae Therapeutic Working Group that high-dose amoxicillin, with or without clavulanate, be used to treat acute otitis media (AOM) addressed concerns about the efficacy of existing therapies against drug-resistant S. pneumoniae. This recommendation relied on pharmacodynamic predictions of concentrations of amoxicillin in middle-ear fluid remaining higher than minimum inhibitory concentrations against intermediately resistant S. pneumoniae for >40% of the dosing interval. OBJECTIVE: This study compared the tolerability and efficacy of cefprozil and high-dose amoxicillin/clavulanate in patients with AOM. METHODS: Patients were randomized to receive 10 days of investigator-blinded oral treatment with either cefprozil suspension (30 mg/kg/d in 2 divided doses) or amoxicillin/clavulanate (45/6.4 mg/kg/d) plus amoxicillin (45 mg/kg/d) in 2 divided doses. The primary efficacy end point was the clinical cure rate 4 to 7 days after the end of treatment. Clinical response by age (6 months-<2 years vs > or =2-7 years), disease severity, and unilateral versus bilateral ear infection was also examined. The primary measures of tolerability were the frequency and severity of adverse events and their relation to study drug. Adverse events were either spontaneously reported or elicited during examination and questioning of the patient. Identified adverse events were coded and recorded using the COSTART (Coding Symbols for Thesaurus of Adverse Reaction Terms) system. RESULTS: Three hundred four children between the ages of 6 months and 7 years with > or =1 sign or symptom of AOM were enrolled in the study, and 303 (150 cefprozil, 153 amoxicillin/clavulanate) were treated. Twenty-three patients in each treatment group were not evaluable; thus, 257 children were included in the analysis of evaluable patients. Clinical cure rates were 87% (110/127) with cefprozil and 89% (116/130) with amoxicillin/clavulanate (95% CI for the difference in cure rate, -10.7% to 4.1%). No between-group differences in efficacy were noted by age, disease severity, or unilateral or bilateral involvement. The overall incidence of drug-related adverse events was significantly lower with cefprozil than with amoxicillin/clavulanate (19% vs 32%, respectively; P = 0.008), as was the incidence of diarrhea (9% vs 19%, respectively; P = 0.021). Adverse events prompted discontinuation of therapy in 4 (3%) cefprozil patients and 8 (5%) amoxicillin/clavulanate patients. CONCLUSIONS: Based on a search of MEDLINE, this study is the first direct comparison of cefprozil versus high-dose amoxicillin/clavulanate. Cefprozil was as effective as high-dose amoxicillin/clavulanate, with a lower incidence of adverse events.

Retropharyngeal abscess: epiglottitis of the new millennium.

From 1993 through 1999, 26 children with retropharyngeal abscess and 2 children with acute epiglottitis were cared for by pediatric otolaryngologists in northern Virginia. Fever, sore throat, dysphagia, refusal to swallow, dysphonia, drooling, and neck extension are common presenting signs and symptoms in acute epiglottitis and in retropharyngeal abscess. Contrast-enhanced computed tomography of the oropharynx was performed in all cases and was the most helpful diagnostic test.

Computed tomography imaging of the maxillary and ethmoid sinuses in children with short-duration purulent rhinorrhea.

INTRODUCTION: Adults with a common cold often have paranasal sinus effusions detected by computed tomographic (CT) scans. There are no comparable data for children. The purpose of this study was to document the sinus CT findings in children with short-duration purulent rhinorrhea. DESIGN: Thirty children, 3 to 12 years of age (median age, 7 years), with purulent rhinorrhea for a mean duration of 5 days (and always less than 9 days) were enrolled in the study. The children were otherwise well. Institutional Review Board (IRB)-approval was obtained before enrollment of the first patient. Informed written consent was obtained from each child's parent. CT imaging of the maxillary and ethmoid sinuses was obtained on the day of the initial visit (occasionally, the following day). Follow-up CT scans were obtained from cooperative children/parents, 3 to 4 weeks later. RESULTS: Opacification or an air/fluid level in the maxillary sinuses was seen in 27 (90%) of 30 study children at study entry. Ethmoid sinuses were not opacified without opacification of a maxillary sinus. Three weeks later, 24 of 27 study children, who had positive CT scans on study entry, improved clinically. Of 17 follow-up CT scans, 10 (58%) normalized, 4 had improvement of bilateral disease, and 3 improved with unilateral disease. None appeared worse than baseline. CONCLUSIONS: Pansinus opacification (ethmoid and maxillary sinuses), on CT scans in children with short-duration purulent nasal drainage was seen in 70% of children. An additional 20% had isolated maxillary sinus effusions (10% had no effusion). Three-week follow-up CT scans on 17 children were normal in 60% and improved (partial clearance) in 40%. In this patient population, the decision to treat with antibiotics should be made on clinical grounds alone.

Nedocromil sodium 2% ophthalmic solution for the treatment of ragweed pollen seasonal allergic conjunctivitis.

PURPOSE: To determine the efficacy and safety of nedocromil sodium 2% ophthalmic solution in the treatment of seasonal allergic conjunctivitis. METHODS: A combined analysis of two multicenter, randomized, comparative, double-masked, placebo-controlled clinical trials involving 261 patients diagnosed with seasonal allergic conjunctivitis was used. Patients were randomly assigned to receive either topical 2% nedocromil sodium or placebo twice daily for eight weeks. Diary card scores and clinician assessments of allergic symptoms were recorded throughout the study; efficacy was determined by comparing symptom severity at the peak pollen period with symptom severity at baseline. Clinician and patient evaluations of treatment effectiveness were used as secondary measurements of efficacy. RESULTS: Patients treated with nedocromil sodium experienced improvement in allergy symptoms, with reductions in the summary symptom score, itch, redness, conjunctival injection, and conjunctival edema significantly (p<0.05) greater than those observed in the patients treated with placebo. Clinicians' and patients' opinions of nedocromil sodium treatment effectiveness were significantly (p<0.02) superior to those of placebo treatment effectiveness. CONCLUSION: Nedocromil sodium is effective in the management of seasonal allergic conjunctivitis.

Characterization of the mouse gene, human promoter and human cDNA of TSCOT reveals strong interspecies homology.

The regulation of gene expression in thymic epithelial cells is critical for T cell development. The mouse thymic epithelial gene Tscot encodes a protein with weak homology to bacterial 12 transmembrane co-transporters. Using competitive reverse transcription-polymerase chain reaction (RT-PCR), we show that low level Tscot expression is detectable in several other tissues. Tscot was mapped to chromosome 4 and was also detected in other mammalian species by Southern blotting. The human cDNA clone showed 77% amino acid identity with the mouse sequence. The highest conservation was in the TM regions and in a small segment of the central cytoplasmic loop. Genomic clones spanning 17164 bases of the Tscot gene revealed four exons with nine of the TM domains encoded in the first exon. The major transcriptional start site in mouse was identified by a primer extension analysis and confirmed by RT-PCR. Comparison of 1.7 kb of the human and mouse promoters identified six conserved possible regulatory elements, one containing a potential binding site for an interferon alpha inducible factor. Finally, as a functional test, 3 kb of the murine promoter was used to create a transgenic mouse that expresses enhanced green fluorescent protein message strongly in the thymus, weakly in the kidney and undetectably in the spleen, liver and heart.

Efficacy and safety of nedocromil sodium 2% ophthalmic solution b.i.d. in the treatment of ragweed seasonal allergic conjunctivitis.

The efficacy and safety of twice-daily nedocromil sodium 2% ophthalmic solution and vehicle were compared in the treatment of ragweed seasonal allergic conjunctivitis. Two separate multicenter, randomized, double-masked, placebo-controlled studies were subjected to a combined analysis. Following a one-week baseline period during the beginning of the ragweed pollen season, 189 patients with seasonal allergic conjunctivitis received either nedocromil sodium or vehicle b.i.d. for eight weeks. Efficacy was evaluated by patient diary cards and clinical eye examinations. Safety was assessed by reports of adverse events. Compared with vehicle, nedocromil sodium produced significantly greater decreases in summary symptom score (p = 0.005), itch (p = 0.005), tearing (p = 0.004), overall eye condition (p = 0.001), and clinician-evaluated conjunctival edema (p = 0.018), and significantly better (p = 0.001), and patient (p = 0.001) opinions of treatment effectiveness at the peak pollen period. Additionally, the superiority of nedocromil sodium compared to vehicle approached statistical significance in redness reduction (p = 0.087) and clinician-evaluated conjunctival injection (p = 0.087). There were no serious treatment-related adverse events in either treatment group. In summary, nedocromil sodium 2% ophthalmic solution b.i.d. was found to be effective and to have a favorable safety profile in the treatment of seasonal allergic conjunctivitis.

Biallelic expression of the IL-2 locus under optimal stimulation conditions.

Recent experiments have suggested that the IL-2 locus is monoallelically expressed. We tested this hypothesis using TCR-transgenic mice carrying one inactivated IL-2 allele. The frequency in single-cell assays of IL-2-producing cells following optimal stimulation by antigen and antigen-presenting cells was equivalent to that from wild-type mice, but the amount of IL-2 produced per cell was twofold less. Similar observations were made by intracellular staining for IL-2, although stimulation in bulk culture was less optimal, showing only a 1.7-fold difference. Importantly, the frequency of responding cells from the heterozygotes was less than from the wild-type mice if the IL-2 assay was performed after only 24 - 30 h of activation, suggesting that the targeted allele could compete with the normal allele early after stimulation and give the misimpression that the heterozygotes had fewer IL-2-producing cells. These data strongly argue that the IL-2 locus can be expressed biallelically under optimum stimulation conditions.

Drug-facilitated sexual assault ('date rape').

In the past few years, drug-facilitated sexual assaults have received widespread media coverage. In addition to alcohol, the most frequently used date-rape drug, flunitrazepam (Rohypnol), a fast-acting benzodiazepine, and gamma-hydroxybutyrate (GHB) and its congeners are among the most popular drugs used for this purpose. The latter drug is easily procured at some gymnasiums, popular bars, discos, and rave clubs, as well as over the Internet. Perpetrators choose these drugs because they act rapidly, produce disinhibition and relaxation of voluntary muscles, and cause the victim to have lasting anterograde amnesia for events that occur under the influence of the drug. Alcoholic beverages potentiate the drug effects. We review several date-rape drugs, provide information on laboratory testing for them, and offer guidelines for preventing drug-facilitated sexual assault.

Medical exclusion of sick children from child care centers: a plea for reconciliation.

BACKGROUND: Policies for excluding ill children from child care can affect parental absenteeism from the workplace and the utilization of pediatric health care resources. METHODS: We surveyed a representative sample of 310 child care centers throughout Virginia to assess policies for excluding children with fever, common upper respiratory tract illnesses, or head lice. RESULTS: Of the 183 center directors (59%) who returned completed surveys, 119 (69%) considered a temperature of 100.0 degrees F to 100.4 degrees F to represent fever, but methods for measuring temperature varied widely. Most centers excluded children with low-grade fever, even in the absence of changes in their behavior. Other low-threshold policies could exclude afebrile children with white nasal or eye discharge and children with hair nits, even after treated with a pediculicidal shampoo. CONCLUSIONS: Exclusion policies among child care centers in Virginia vary widely and often are inconsistent with current standards of medical practice. More uniform implementation of exclusion policies established by national consensus panels of experts is needed to reduce unnecessary exclusion of children from child care centers.

Antibiotic rashes in children: a survey in a private practice setting.

OBJECTIVE: To document the frequency and severity of various types of rashes seen with commonly used oral antibiotics in the pediatric outpatient setting. DESIGN: A retrospective review of 5923 patient records at a pediatric office. SETTING: A private group pediatric practice in northern Virginia with about 12,000 registered active patients. PATIENTS AND METHODS: Approximately 50% of the clinic medical records were reviewed. All children (defined as those aged 0-18 years in this study) identified on their medical records as having developed a rash following treatment with 1 or more of the commonly used oral antibiotics were included in the study. For further validation, a questionnaire about parental recollection of description of rash, other associated symptoms, physician verification, and outcome was mailed to families with children designated as being allergic to an antibiotic. RESULTS: On a prescription basis, significantly more rashes were documented for cefaclor (4.79%) compared with penicillins (2.72%), sulfonamides (3. 46%), and other cephalosporins (1.04%). Based on the number of patients for whom each group of antibiotic was prescribed, the documented frequencies of rashes were 12.3%, 7.4%, 8.5%, and 2.6% for cefaclor, penicillins, sulfonamides, and other cephalosporins, respectively. None of the children had rashes severe enough to require hospitalization. CONCLUSIONS: In a review of almost 6000 records in a private pediatric primary care setting, rashes occurred in 7.3% of children who were given the commonly used oral antibiotics. Significantly more rashes were documented with cefaclor use than with use of any of the other oral antibiotics.

Epithelial cell-specific laminin 5 is required for survival of early thymocytes.

The gene LamC2 encoding the gamma2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the alpha3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44-subsets of the CD4-CD8- double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin alpha3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.

Acute mastoiditis in children: an increase in frequency in Northern Virginia.

BACKGROUND: Acute mastoiditis is reported to occur 2 or 3 times annually in the largest children's hospitals. We encountered an average of 1 case annually at our hospital from 1986 to 1991. During an 8-year period ending October 31, 1999, 22 patients were diagnosed and treated at our hospital. Of these, 17 presented during the last 34 months. METHODS: Retrospective chart review from office and hospital records of children from infancy to age 12 years with a discharge diagnosis of acute mastoiditis who were treated from 1992 through 1999. RESULTS: All children were referred to one of the two pediatric otolaryngologists in our community because of forward protrusion of the auricle and retroauricular cellulitis. Eleven (50%) were <14 months old. Ninety-five percent had a concomitant ipsilateral, inflamed, bulging, immobile eardrum. Computerized tomographic imaging, performed on all patients, revealed universal cortical destruction, subperiosteal abscess or bone destruction in four and dural venous thrombosis in two. Mastoidectomy was necessary for eight children (36%) because of complications of mastoiditis (n = 4) or for failure to improve with antibiotics and myringotomy drainage (n = 4). Streptococcus pneumoniae or Streptococcus pyogenes was recovered from 10 of 17 children (59%) from whom cultures were obtained. CONCLUSION: Cases of acute mastoiditis have markedly increased in our suburban children's hospital. The disease was most common during infancy. Serious complications of mastoiditis occurred in four (18%) of the children in this series.

Host resistance and immune deviation in pigeon cytochrome c T-cell receptor transgenic mice infected with Toxoplasma gondii.

Resistance to Toxoplasma gondii has been shown to be mediated by gamma interferon (IFN-gamma) produced by NK, CD4(+), and CD8(+) T cells. While studies of SCID mice have implicated NK cells as the source of the cytokine in acute infection, several lines of evidence suggest that IFN-gamma production by CD4(+) T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T-cell receptor (TCR)-dependent stimulation by the parasite, we have examined the resistance to T. gondii infection of pigeon cytochrome c transgenic (PCC-Tg) Rag-2(-/-) mice in which all CD4(+) T lymphocytes are unreactive with the protozoan. When inoculated with the ME49 strain, PCC-Tg animals exhibited only temporary control of acute infection and succumbed by day 17. Intracellular cytokine staining by flow cytometry revealed that, in contrast to infected nontransgenic controls, infected PCC-Tg animals failed to develop IFN-gamma-producing CD4(+) T cells. Moreover, the CD4(+) lymphocytes from these mice showed no evidence of activation as judged by lack of upregulated expression of CD44 or CD69. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses measured after in vitro antigen restimulation displayed a strong Th1 bias which was shown to be dependent on endogenous interleukin 12 (IL-12). The above findings argue that, while T. gondii-induced IL-12 cannot trigger IFN-gamma production by CD4(+) T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against nonparasite antigens, an effect that may explain the immunostimulatory properties of T. gondii infection.

A putative 12 transmembrane domain cotransporter expressed in thymic cortical epithelial cells.

We have isolated a full-length cDNA clone (thymic stromal origin (TSO)-1C12) from a SCID thymus library using a probe from a PCR-based subtractive library enriched for sequences from fetal thymic stromal cells. TSO-1C12 mRNA is expressed mainly in the thymic cortex and is highly enriched in SCID thymus. Expression per cell is highest during fetal thymus development and decreases after day 16. Antipeptide Abs immunoprecipitated a hydrophobic, plasma membrane glycoprotein (thymic stromal cotransporter, TSCOT) whose translated sequence has weak homology to bacterial antiporters and mammalian cation cotransporters with 12 transmembrane domains. TSCOT represents a new member of this superfamily that is highly expressed in thymic cortical epithelial cells.