Search

To assess the effectiveness and outcomes of a targeted cytomegalovirus (CMV) testing protocol.Retrospective chart review.Tertiary care institution.Targeted screening for CMV in children who did not pass the newborn hearing screening (NHS) was introduced in July 2015 for the level 2 and 3 nurseries at our hospital. In January 2016, this policy was extended to include all nurseries. Retrospective chart review was performed for all newborns who did not pass their NHS between 2013 and 2020. CMV testing rates and related outcomes were compared before and after rollout.A total of 891 charts were reviewed for infants who did not pass their NHS: 530 (60%) had CMV testing, of which 8 (1.5%) tested positive. Three cases were detected prior to and 5 occurred after initiation of targeted screening. Six CMV+ infants demonstrated hearing loss on confirmatory auditory brainstem response, all of whom began treatment with oral valganciclovir. Hearing remained stable in 3 patients, progressed in 2, and improved in 1. The rate of CMV testing in children who did not pass their NHS increased from 14% to 88% after full implementation of targeted screening (Targeted screening is a feasible and effective method to identify CMV+ infants early in life. Implementation of a targeted screening program for CMV in children who do not pass the NHS resulted in significantly higher rates of CMV testing and earlier referral to infectious disease.

Background: Inflammation and immune dysregulation have been implicated in the pathogenesis of pediatric-onset obsessive-compulsive disorder (OCD) and tic disorders such as Tourette syndrome (TS). Though few replicated studies have identified markers of immune dysfunction in this population, preliminary studies suggest that serum immunoglobulin A (IgA) concentrations may be abnormal in these children with these disorders. Methods: This observational retrospective cohort study, conducted using electronic health records (EHRs), identified 206 children with pediatric-onset OCD and 1024 adults diagnosed with OCD who also had testing for serum levels of IgA. IgA deficiency and serum IgA levels in pediatric OCD were compared with IgA levels from children diagnosed with autism spectrum disorders (ASD; n = 524), tic disorders (n = 157), attention-deficit/hyperactivity disorder (ADHD; n = 534), anxiety disorders (n = 1206), and celiac disease, a condition associated with IgA deficiency (n = 624). Results: Compared with ASD and anxiety disorder cohorts, the pediatric OCD cohort displayed a significantly higher likelihood of IgA deficiency (OR = 1.93; 95% CI = 1.18–3.16, and OR = 1.98; 95% CI = 1.28–3.06, respectively), though no difference was observed between pediatric OCD and TS cohorts. Furthermore, the pediatric OCD cohort displayed similar rates of IgA deficiency and serum IgA levels when compared with the celiac disease cohort. The pediatric OCD cohort also displayed the highest percentage of IgA deficiency (15%,) when compared with TS (14%), celiac disease (14%), ADHD (13%), ASD (8%), and anxiety disorder (8%) cohorts. When segregated by sex, boys with OCD displayed a significantly higher likelihood of IgA deficiency when compared with all comparison cohorts except for celiac disease and tic disorders; no significant difference in IgA deficiency was observed between female cohorts. Pediatric OCD subjects also displayed significantly lower adjusted serum IgA levels than the ASD and anxiety disorder cohorts. Adults with OCD were also significantly less likely than children with OCD to display IgA deficiency (OR = 2.71; 95% CI = 1.71–4.28). When compared with children with celiac disease, no significant difference in IgA levels or rates of IgA deficiency were observed in the pediatric OCD cohort. Conclusions: We provide further evidence of IgA abnormalities in pediatric-onset OCD. These results require further investigation to determine if these abnormalities impact the clinical course of OCD in children.

Background Massachusetts began newborn screening (NBS) for severe combined immunodeficiency (SCID) using measurement of T-cell receptor excision circles (TRECs) from dried blood spots. Objective We describe developments and outcomes from the first 10 years of this program (February 1, 2009, to January 31, 2019). Methods TREC values, diagnostic, and outcome data from all patients screened for SCID were evaluated. Results NBS of 720,038 infants prompted immunologic evaluation of 237 (0.03%). Of 237, 9 were diagnosed with SCID/leaky SCID (4% of referrals vs 0.001% general population). Another 7 were diagnosed with other combined immunodeficiencies, and 3 with athymia. SCID/leaky SCID incidence was approximately 1 in 80,000, whereas approximately 1 in 51,000 had severe T-cell lymphopenia for which definitive treatment was indicated. All patients with SCID/leaky SCID underwent hematopoietic cell transplant or gene therapy with 100% survival. One patient with athymia underwent successful thymus transplant. No known cases of SCID were missed. Compared with outcomes from the 10 years before SCID NBS, survival trended higher (9 of 9 vs 4 of 7), likely due to a lower rate of infection before treatment. Conclusions Our data support a single NBS testing-and-referral algorithm for all gestational ages. Despite lower median TREC values in premature infants, the majority for all ages are well above the TREC cutoff and the algorithm, which selects urgent (undetectable TREC) and repeatedly abnormal TREC values, minimizes referral. We also found that low naive T-cell percentage is associated with a higher risk of SCID/CID, demonstrating the utility of memory/naive T-cell phenotyping as part of follow-up flow cytometry.

Objectives: Our objective is to highlight the growing problem of multi-drug resistant gram negative bacterial (MDRGNB) species in burn units and demonstrate strategies for successful management. Methods: Using a concurrent data collection, a 20-year experience with MDRGNB in a pediatric burn unit was reviewed. Results: 220 (6.5%) of 3,359 children admitted for management of acute burns or open wounds had MDRGNB recovered. The children had an average age of 8.8 ± 0.3 years and average burn size of 42.9 ± 22.8 percent. The incidence of MDRGNB increased over the 20 years of the study from 2.5% of admissions in the first decade to 8.6% in the second. 90% (198) were found colonized with MDRGNB on admission on transfer from other facilities. MDRGNB sepsis (blood recovery) occurred in 80 (36.4%), MDRGNB invasive wound infection in 46 (20.9%), MDRGNB pneumonia in 19 (8.6%), and MDRGNB UTI in 46 (20.9%) of the children. There were 14 (6.4%) fatalities, the cause of death being MDRGNB sepsis in all 14 children. However the large majority of the children 206 (93.6%) survived despite their MDRGNB infection. Conclusions: MDRGNB may be transferred into burn units during transfers of patients with complex wounds and can be the cause of life threatening infections. The number of children arriving with MDRGNB organisms has steadily increased over time. MDRGNB infected patients can be successfully managed with wound excision and closure supported by topical mafenide acetate and administration of the limited antibiotics to which the organisms are sensitive, particularly Colistin. Strict infection control practices will limit cross infection with these difficult organisms. Keywords: Burns, Children, Resistant bacteria, Sepsis, Drug resistance, Multiple drug resistance

OBJECTIVES: Emergency department (ED) utilization is a major driver of cost. Specialist physicians have an important role in addressing ED utilization, especially at tertiary medical centers that treat highly specialized patients. We analyzed if reporting of ED utilization to pediatric specialist physicians can decrease ED visits. METHODS: Physicians within pediatric neurology, hematology and oncology, infectious diseases, and pulmonary divisions received their ED use reports. By using control charts, we examined if this intervention decreased the rate of ED utilization. RESULTS: Overall, for the 4 divisions, specialty-related ED utilization decreased significantly during all hours, weekdays, and office hours. This was in the setting of ED utilization increasing for all diagnoses ED visits. Pediatric ED volume did not change during the study period. CONCLUSIONS: Physician-level reporting of ED utilization was associated with a reduction in ED use by patients managed by our pediatric specialists.

A previously healthy 19-year-old college student was transferred to this hospital with fever, hypotension, and pain in the right elbow and left ankle. Eleven days earlier, sore throat and fatigue had developed, and a diagnosis of infectious mononucleosis was made; 1 week later, corticosteroids were prescribed because of severe pharyngitis. On the day of admission, the patient awoke with severe joint pain. Laboratory studies on admission showed leukocytosis and anemia. The next day, the result of a diagnostic test was reported.

RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.

On May 23 and 24, 2013, the First PANS Consensus Conference was convened at Stanford University, calling together a geographically diverse group of clinicians and researchers from complementary fields of pediatrics: General and developmental pediatrics, infectious diseases, immunology, rheumatology, neurology, and child psychiatry. Participants were academicians with clinical and research interests in pediatric autoimmune neuropsychiatric disorder associated with streptococcus (PANDAS) in youth, and the larger category of pediatric acute-onset neuropsychiatric syndrome (PANS). The goals were to clarify the diagnostic boundaries of PANS, to develop systematic strategies for evaluation of suspected PANS cases, and to set forth the most urgently needed studies in this field. Presented here is a consensus statement proposing recommendations for the diagnostic evaluation of youth presenting with PANS.

The cytotoxic T lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-strand nicking is observed instead of oligonucleosomal fragmentation. Granzyme A is a specific tryptase that concentrates in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by the caspase activator granzyme B. Here we show that granzyme A treatment of isolated nuclei enhances DNA accessibility to exogenous endonucleases. In vitro and after cell loading with perforin, GrnA completely degrades histone H1 and cleaves core histones into approximately 16-kDa fragments. Histone digestion provides a mechanism for unfolding compacted chromatin and facilitating endogenous DNase access to DNA during T cell and natural killer cell granule-mediated apoptosis.

Many people harbor herpes simplex virus, often with a known history of “cold sores”. During the relatively immunosuppressed state associated with a serious burn, recrudescence of such infections can occur. We report four adults and two children who developed severe herpetic ulceration, over the face and neck in five patients and in a partial thickness wound in one patient. Herpetic infection was diagnosed by culture and direct immunofluorescence testing and treatment was immediately instituted with systemic and topical Acylovir® (Zovirax, Glaxo Wellcome). Ulceration healed under treatment and did not leave visible scarring in any of the patients. Although these infections are rapidly progressive, they respond to prompt treatment with antiviral chemotherapy. Rapidly progressive vesicles and ulceration appearing on the face or in the wounds of burn patients should prompt immediate evaluation for herpetic infection.

We have cloned a novel pancreatic beta cell and neuroendocrine cell-specific calcium-binding protein termed secretagogin. The cDNA obtained by immunoscreening a human pancreatic cDNA library using the recently described murine monoclonal antibody D24 contains an open reading frame of 828 base pairs. This codes for a cytoplasmic protein with six putative EF finger hand calcium-binding motifs. The gene could be localized to chromosome 6 by alignment with GenBank genomic sequence data. Northern blot analysis demonstrated abundant expression of this protein in the pancreas and to a lesser extent in the thyroid, adrenal medulla, and cortex. In addition it was expressed in scant quantity in the gastrointestinal tract (stomach, small intestine, and colon). Thyroid tissue expression of secretagogin was restricted to C-cells. Using a sandwich capture enzyme-linked immunosorbent assay with a detection limit of 6.5 pg/ml, considerable amounts of constitutively secreted protein could be measured in tissue culture supernatants of stably transfected RIN-5F and dog insulinoma (INS-H1) cell clones; however, in stably transfected Jurkat cells, the protein was only secreted upon CD3 stimulation. Functional analysis of transfected cell lines expressing secretagogin revealed an influence on calcium flux and cell proliferation. In RIN-5F cells, the antiproliferative effect is possibly due to secretagogin-triggered down-regulation of substance P transcription.

Purpose To document outcomes of management of juvenile mandibular chronic osteomyelitis (JMCO) using a standardized treatment protocol including open biopsy, decortication, microbial culture, and long-term antibiotic therapy. Materials and Methods This was a retrospective case study of pediatric patients with JMCO treated at Massachusetts General Hospital for Children from 1996 through 2014. Inclusion criteria included age younger than 18 years, diagnosis of JMCO, management by the protocol, adequate clinical and radiographic data in the record, and follow-up of at least 1 year after initial treatment. Inpatient and outpatient records were reviewed for demographics, clinical and radiographic findings, and histologic and laboratory evaluations. The predictor variable was the standardized treatment protocol and the primary outcome variables were disease status at end of treatment and complications of treatment. Results Twenty patients (mean age at onset, 10.7 yr; range, 3 to 14 yr) were treated, 12 (11 girls) of whom met the inclusion criteria. Management of all patients consisted of biopsy (extraoral when feasible, n = 9; intraoral, n = 3), decortication, cultures, and long-term antibiotic therapy. Nonsteroidal anti-inflammatory drugs (NSAIDs) were administered only as needed for pain control. There was complete resolution of osteomyelitis with no recurrence in 7 of 12 patients (mean follow-up, 4.3 yr; range, 1 to 11 yr). Five patients had partial responses, with decreased frequency and severity of disease recurrence. These were well controlled with short courses of antibiotics (4 to 12 weeks) with NSAIDs only as needed for pain control (mean follow-up, 1.4 yr; range, 1 to 3 yr). There were no major complications related to antibiotic therapy. Minor complications included rash (n = 2), nausea and vomiting (n = 1), and vaginal candidiasis (n = 1). Conclusion The results of this study indicate that 58.3% of patients were cured and had no recurrent symptoms (mean follow-up, 4.3 yr). The remaining patients continue on intermittent treatment with antibiotics for recurrent episodes of swelling and pain. Favorable responses to antibiotic therapy support the hypothesis that JMCO is an infectious disease and that negative cultures might represent a failure of standard culture techniques to isolate the responsible organisms.

CD8+ lymphocytes are believed to be important in host defence against the human immunodeficiency virus (HIV)-1, inhibiting HIV-1 replication through both cytolytic and non-cytolytic pathways. The cytolytic pathway involves calcium-dependent exocytosis of perforin and granzyme proteases, as well as Fas-mediated programmed cell death, whereas the noncytolytic pathway involves the release of chemokines that prevent viral entry. Using granzyme A as a marker of cytolytic granule proteins, and macrophage inflammatory protein (MIP)-1alpha and RANTES as markers of HIV-1 inhibitory chemokines, we show that these two very different mediators of viral inhibition are both localized in the cytolytic granules of HIV-1-specific CD8+ cytotoxic T lymphocytes (CTL). Following antigen-specific activation, these mediators are secreted together, facilitating both lysis of virion-producing cells and the inhibition of free virus. In addition, RANTES, MIP-1alpha and MIP-1beta are secreted by CTL as a macromolecular complex containing sulphated proteoglycans. This association appears to have a functional significance, because heparan sulphate facilitates RANTES inhibition of HIV-1 infection of monocytes.

Members of the CED-3/interleukin-1beta-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. We report here that caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32-33 kDa and 14 kDa, which are then further processed into 18- and 12-kDa active subunits. Up to 50 microM N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (50 microM) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (50 nM), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 microM DEVD-CHO. Our results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function.

Viruses, enteric bacteria and parasites can all produce similar syndromes of acute enteritis, although the pathophysiology and molecular pathogenesis may vary widely. The severity of acute enteritis' varies greatly, and only a small fraction of cases undergo medical evaluation. There are over 200 000 000 episodes of acute enteritis annually in the USA, of which approximately 75 000 000 are foodborne. Fewer than 20% of estimated cases have a known etiology. Nearly half of the more than 4 000 000 cases of known foodborne bacterial enteritis are attributed to Campylobacter infections. The FoodNet active population-based surveillance system has demonstrated geographic variation in the incidence of Campylobacter infection, and generally declining incidence since 1996. Campylobacter jejuni infections vary in severity and duration. Antimicrobial therapy, especially if administered early, may hasten clinical resolution by 2–3 days. Therapy is generally restricted to individuals with moderate-to-severe disease and high-risk individuals (underlying immunodeficiency, chronic illness,,extremes of age, etc.). Approximately 10–15 % of C. jejuni isolates are fluoroquinolone resistant, although most strains are macrolide susceptible. Several acute intra-abdominal as well as extraintestinal complications can occur following C. jejuni enteritis, although bacteremia and metastatic infection are rare, except in high-risk patients. Post-infectious syndromes include post-dysenteric bowel dysfunction, the less frequent but more severe reactive arthritis (1%), and Guillain-Barre syndrome (0.1%). The recent demonstration that antibiotic administration for Escherichia coli 015TH7 hemorrhagic enteritis increases the risk of hemolyticuremic syndrome 5–10-fold forces reconsideration of empirical broad-spectrum antibiotic therapy for acute hemorrhagic enteritis, and supports narrow-spectrum erythromycin therapy for acute enteritis.

During acute Lyme disease, bacteria can disseminate to the central nervous system (CNS), leading to the development of meningitis and other neurologic symptoms. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing a deep view into the proteome for patients diagnosed with early disseminated Lyme disease and CSF inflammation. Additionally, we analyzed individual patient samples and quantified differences in protein abundance employing label-free quantitative mass spectrometry-based methods. We identified 108 proteins that differ significantly in abundance in patients with acute Lyme disease from controls. Comparison between infected patients and control subjects revealed differences in proteins in the CSF associated with cell death localized to brain synapses and others that likely originate from brain parenchyma.

The Centers for Disease Control and Prevention guidelines for prevention of intravascular catheter-related infections suggest that antimicrobial-coated catheters can decrease the risk of developing catheter-related bloodstream infection in a variety of adult patient populations. There are limited data on their efficacy in the pediatric population, particularly among children with burn injuries. A study was conducted at Shriners Hospitals for Children®, Boston, to determine whether minocycline/rifampin (MR)-coated catheters could decrease the incidence of catheter-associated bloodstream infection (CABSI) in a pediatric burn population. A historical control group included all patients with double- or triple-lumen catheters inserted in the 18-month period from January 2006 to June 2007. The study group included all patients with MR antimicrobial double- or triple-lumen catheters inserted in the subsequent 18-month period, July 2007 to December 2008. Data collected included name, age, date of burn/injury, date of admission, percent TBSA area burn injury or other diagnosis, catheter site (subclavian, internal jugular, or femoral), method of insertion (new percutaneous stick or guidewire), type of catheter (double or triple lumen), date inserted, duration of catheter placement (days), and positive blood cultures recovered while the central venous catheter was in place. CABSI was defined using the Centers for Disease Control and Prevention definition of laboratory-confirmed bloodstream infection. There were a total of 66 patients with 252 catheters (1780 catheter days) in the control group and 75 patients with 263 catheters (1633 catheter days) in the study group. Age, percent burn injury, catheter site, and method of insertion were not statistically different between the two groups. The percentage of infected catheters and the rate of infection were significantly different for the two groups, with the MR antimicrobial catheters only half as likely to become infected. In a subset of these patients with catheters in place for more than 4 days, the percentage of infected catheters and rate of infection were also significantly different with results similar to those in the entire group. MR antimicrobial-coated catheters significantly reduced the incidence of CABSI in this pediatric burn population compared with noncoated catheters.

Background Most infants with congenital Toxoplasma gondii infection have no symptoms at birth, but many will have retinal disease or neurologic abnormalities later in life. Early detection and treatment of congenital toxoplasmosis may reduce these sequelae. Methods In Massachusetts since January 1986, and in New Hampshire since July 1988, newborns have been screened for intrauterine infection with T. gondii by means of an IgM capture immunoassay of blood specimens routinely collected for screening for metabolic disorders. Congenital infection is confirmed by assays for specific IgG and IgM antibodies in serum from infants and their mothers. For this study, infants with serologic evidence of infection underwent extensive clinical evaluation and received one year of treatment. Results Through June 1992, 100 of 635,000 infants tested had positive screening tests. Congenital infection was confirmed in 52 infants, 50 of whom were identified only through neonatal screening and not through initial clinical exami...

Presentation of Case A 51-year-old woman was admitted to the hospital with diffuse aches and mediastinal lymphadenopathy. The patient was well until 15 months earlier, when she began to experience pain in the shoulders, right arm, and legs with the onset of cold weather. She was seen in a hospital in her native Ethiopia, where injections were administered and oral medications were prescribed, without improvement. Six months before admission sweats developed and continued; the patient was uncertain about the presence of fever. One month later she found that her myalgias and arthralgias interfered with her sleep and were accompanied by . . .

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram-negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti-lipid A mAbs function by neutralizing the toxic effects of LPS.

The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells.

RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.

Background: Historically, group A beta-hemolytic streptococci (GAS) burn wound infection has been a major source of morbidity and mortality in burn patients and has prompted the prophylactic administration of antibiotics to children with burns. Wound monitoring, surveillance cultures, and early excision of deep wounds may have changed this. Our objective in this project was to determine the efficacy of routine antibiotic prophylaxis in the era of early excision and closure of deep burn wounds. Methods: Two cohorts of burned children were compared: all children admitted during calendar years 1992 through 1994 (group 1) and during calendar years 1995 through 1997 (group 2). All group 1 children received routine GAS antibiotic prophylaxis. Only those group 2 children with documented positive admission or surveillance cultures for GAS were treated. Results: There were 511 children in group 1 and 406 children in group 2. They were well matched for age (4.7 ± 0.21 years vs. 5.3 ± 0.26 years, p = 0.06) and burn size (11.0% ± 0.7% vs. 12.4% ± 0.8%,p = 0.18). GAS species were recovered at admission or during hospitalization from 11 (2.6%) of group 1 children and 18 (4.4%) of group 2 children (p = 0.05), indicating a marginally higher rate of carriage in group 2. Nevertheless, in group 1 there were three (0.6%) who developed GAS wound infection and in group 2 there were four (0.98%,p = 0.71). The incidence of GAS infection in those patients with positive admission cultures was three (27%) of group 1 and four (22%) of group 2. No child developed fulminant GAS infection. Conclusion: Routine antibiotic prophylaxis of burn wounds in children in not effective in further reducing a low baseline incidence of GAS wound infection if admission screening by culture is used to identify those children who carry the organism and early excision of deep burns is practiced.

Human haptoglobin (Hp) is synthesized at hepatic and extrahepatic sites as an acute-phase reactant protein (APP). We investigated the effects of Hp on granulocyte function. The chemotaxis of freshly isolated human granulocytes and differentiated HL-60 cells in response to the bacterial tripeptide, f-met-leu-phe, was inhibited in the presence of a physiological concentration of Hp, but chemotaxis in the presence of the proinflammatory cytokine interleukin-8 (IL-8) was not inhibited. Phagocytosis of viable Escherichia coli, as well as fluoresceinated nonviable E. coli, was inhibited. Hp also reduced granulocyte intracellular bactericidal activity against E. coli. The observed inhibitory effects of Hp on granulocyte function are similar to those reported for C-reactive protein and suggest that APPs dampen the acute inflammatory response.

Introduction: The fi rst step in the pathogenesis of most skin and soft tissue infections is the colonization of the host by a potential pathogen or pathogens. Staphylococcus aureus has long been an important pathogen, but the dissemination of community-associated methicillin-resistant S. aureus (MRSA) over the past decade, superimposed on the gradually increasing carriage rates of hospital-associated MRSA, have made beta-lactam–resistant S. aureus isolates the dominant etiology of skin and soft tissue infections in many locations across the United States [ 1 ]. The costs of established MRSA infection with regard to prolonged hospital stay, increased mortality, and increased expense have been widely reported [ 2 ], and it is only natural that growing attention has been focused on preventing MRSA infection. Although the anterior nares are the most commonly reported site of MRSA carriage, MRSA may persist in additional sites (eg, skin, chronic wounds, and perianal/rectal carriage) in some individuals or may reside exclusively in sites other than the nares in others. The dynamic aspects of S. aureus carriage are complex: most individuals are transiently colonized for varying intervals, some are persistently colonized at one or more sites when studied longitudinally and a modest fraction of individuals remain persistently culture negative for S. aureus colonization. Several interventional studies have explored the eradication of MRSA carriage. Eradication strategies have tended to use topical agents directed at nares and/or skin decontamination or systemic antibiotics. In general, these studies have been limited by enrollment size, duration of monitoring, and inherent study limitations regarding the distinction in culture-positive patients between decontamination failures and effective eradication followed by acquisition of a distinct MRSA strain.

Background: Nosocomial infections (NI) are believed to occur more commonly in patients with burns than in patients undergoing surgery, but benchmark rates have not been well described, and widely accepted definitions of NI in patients with burns are not available. We present a clinically useful set of definitions for NI for the pediatric burn population and provide benchmark infection rates for NI at selected sites. Methods: Centers for Disease Control and Prevention definitions were modified to more accurately describe nosocomial burn infection and secondary bloodstream infections (BSI) in the burn population. A surveillance system was developed and included calculation of NI rates by 1000 patient or device days, stratified into one of three risk groups ( 60% burn injury). All patients with acute burns admitted from January 1990 to December 1991 were included, and NI rates were calculated for burn infection, primary and secondary BSI, ventilator-related pneumonia and urinary catheter-related urinary tract infection (UTI). Results: Overall 12.5% of patients with central venous catheters had development of primary BSI for a rate of 4.91000 central venous catheter-days. Incidence of secondary BSI was 5.8% of patients for a rate of 5.31000 patient-days. Incidence of burn infection was 10.1% of patients for a rate of 5.61000 patient-days. Incidence of ventilator-related pneumonia was 17.5% of patients for a rate of 11.41000 ventilator-days. Incidence of urinary catheter-related UTI was 17.9% of patients, for a rate of 13.21000 urinary catheter-days. When rates were stratified by risk groups, incidence increased with increasing burn size for secondary BSI ( p ≤ 0.0001) and urinary catheter-related UTI ( p = 0.08), although rates based on number of patient-days or device-days more accurately reflected risk of infection over time. Conclusions: Infection remains a cause of significant morbidity and death for patients with burns. The definitions and benchmark rates reported here may be useful in evaluation of NI surveillance strategies and calculation of infection rates, which could then be used to evaluate current treatment modalities and improve outcomes for the burn population.

Recurrent infections often reflect underlying abnormalities, either anatomic or immunologic. In this paper we review how to recognize the underlying disorders in a variety of recurrent infections.

Objective. Despite dramatic reductions in perinatal human immunodeficiency virus (HIV) transmission in the United States, obstacles to perinatal HIV prevention that include lack of prenatal care; failure to test pregnant women for HIV before delivery; and lack of prenatal, intrapartum, or neonatal antiretroviral (ARV) use remain. The objective of this study was to describe trends in perinatal HIV prevention methods, perinatal transmission rates, and the contribution of missed opportunities for perinatal HIV prevention to perinatal HIV infection. Methods. We analyzed data obtained from infant medical records on 4755 HIV-exposed singleton deliveries in 1996–2000, from 6 US sites that participate in the Centers for Disease Control and Prevention’s Pediatric Spectrum of HIV Disease Project. HIV-exposed deliveries refer to deliveries in which the mother was known to have HIV infection during the pregnancy. Results. Of the 4287 women with data on prenatal care, 92% had prenatal care. From 1996 to 2000, among the 3925 women with prenatal care, 92% had an HIV test before delivery; the use of prenatal zidovudine (ZDV) alone decreased from 71% to 9%, and the use of prenatal ZDV with other ARVs increased from 6% to 70%. Complete data on maternal and neonatal ARVs were available for 3284 deliveries. Perinatal HIV transmission was 3% in 1651 deliveries with prenatal ZDV in combination with other ARVs, intrapartum ZDV, and neonatal ZDV; 6% in 1111 deliveries with prenatal, intrapartum, and neonatal ZDV alone; 8% in 152 deliveries with intrapartum and neonatal ZDV alone; 14% of 73 deliveries with neonatal ZDV only started within 24 hours of birth; and 20% in 297 deliveries with no prenatal, intrapartum, and neonatal ARVs. Complete data on prenatal events were available in 328 HIV-infected and 3258 HIV-uninfected infants. A total of 56% of mothers of HIV-infected infants had missed opportunities for perinatal HIV prevention versus 16% of mothers of HIV-uninfected infants. Forty-four percent of the infected infants were born to mothers who had prenatal care, a prenatal HIV diagnosis, and documented prenatal ARV therapy. Seventeen percent of women with reported illicit drug use had no prenatal care versus 3% of women with no reported drug use. In a multivariate analysis, maternal illicit drug use was significantly associated with lack of prenatal care. In a multivariate analysis, year of infant birth and the combination of lack of maternal HIV testing before delivery and lack of prenatal antiretroviral therapies were significantly associated with perinatal HIV transmission. Conclusions. Missed opportunities for perinatal HIV prevention contributed to more than half of the cases of HIV-infected infants. Prenatal care and HIV testing before delivery are major opportunities for perinatal HIV prevention. Illicit drug use was highly associated with lack of prenatal care, and lack of HIV testing before delivery was highly associated with perinatal HIV transmission.

Background : Control of methicillin-resistant Staphylococcus aureus (MRSA) is particularly difficult in burn units, which are often cited as sources of hospital-wide MRSA outbreaks. We developed a successful MRSA control program and document here its apparent effectiveness in controlling MRSA transmission in a pediatric burn unit. Methods : An MRSA control program that included surveillance culturing, clinician feedback, flexible, site-specific isolation, and a list of known carriers was consistently applied in a pediatric burn unit through a 7-year period. Microbiology reports of MRSA isolates from patients and environmental surfaces and records of all patients from whom MRSA was isolated were reviewed. Results : During calendar years 1985 through 1991, a total of 991 acutely burned children were admitted to the Boston unit of the Shriners Burns Institute. Forty MRSA cases (4%) were identified. One patient both had MRSA at admission and met our criteria for nosocomial MRSA. Of the remaining 39 patients, 11 had MRSA at admission and 28 had nosocomial MRSA. There were 17 wound infections, two cases of pneumonia, and two bloodstream infections. No deaths were attributed to MRSA sepsis. Conclusion : An MRSA control program including surveillance culturing, clinician feedback, flexible, site-specific isolation, and a list of known carriers is associated with a low rate of nosocomial MRSA in a pediatric burn unit.

Despite considerable information regarding the structure and expression of granzymes, the serine proteases that reside in the cytotoxic granules of cytotoxic T lymphocytes (CTL), little is known about their physiologic substrates or their role in the economy of these effector cells. From a conceptual framework, such proteases may act on the effector cells themselves (“cis” action), on the target cells (“trans”), or in a more nonspecific fashion; current information remains sketchy and controversial. In this chapter, the functional properties and possible physiologic roles of CTL granzymes will be reviewed, with particular reference to granzyme A.

Treatment of cytomegalovirus (CMV) infections has assumed increasing importance in the care of immunocompromised patients, particularly allograft recipients and patients with symptomatic infection with the human immunodeficiency virus. The crucial role of T cells in the host defense against CMV may be inferred by the expression of life-threatening CMV disease in these restricted patient subpopulations. However, detailed understanding of the pathogenesis of CMV disease in the compromised host is lacking, and the mechanisms by which T cells confer protection are unknown. Insight into the pathogenesis of CMV in these patients is crucial for the development of rational therapeutic strategies. A considerable body of data has been generated to explore the role of T cell-mediated immunity in a number of model viral infections as well as in experimental CMV infections in both normal and immunocompromised mice. This experience will be critically reviewed to clarify the often conflicting observations regarding a protective or, conversely, a pathogenic role for T cells in producing CMV disease.

Clones of cytotoxic T lymphocytes (CTL) differ in their specific reactivity with diverse target cell antigens. To learn about the uniqueness of individual CTL clones we injected rats and mice with cloned CTL in an effort to prepare clone-specific antisera and to analyze the CTL surface molecules that were immunoprecipitated by these antisera. Three clones were studied. They were all derived from BALB.B mice and were specific for antigens encoded by the major histocompatibility complex of the H-2d haplotype. Antisera raised in rats against individual clones contained antibodies to lymphocyte function-associated antigen type 1 (LFA-1) and inhibited the cytotoxic activity of all of the clones. In contrast, BALB/c and BALB.K mice injected with individual clones consistently yielded alloantisera that were clone specific in their ability to inhibit CTL-mediated lysis of target cells (P815). In addition, these alloantisera immunoprecipitated from extracts of 125I-radiolabeled CTL a disulfide-bonded dimer consisting of approximately equal to 45-kilodalton subunits. This dimer resembles the putative T-cell antigen-recognition receptor recently identified in several laboratories. The alloantisera also immunoprecipitated CTL surface molecules that were associated with beta 2-microglobulin and that differed in apparent molecular mass (37-38 kilodaltons) in different clones.

The mechanisms that enable cytotoxic T lymphocytes (Tc cells) to destroy target cells are only vaguely understood. However, recent studies have identified in Tc cells1 and natural killer cells2 cytoplasmic granules that contain perform, a cytolytic protein that resembles the ninth component of complement (C9)3. Antigen-specific lysis of target cells, traditionally ascribed solely to Tc cells, has now also been demonstrated in some T-helper cell (Th cell) lines, referred to here as T helper–killer or Th/c cells4–6. We recently found a novel serine esterase that is present at greatly elevated levels in cloned murine Tc cell lines and one Th/c cell line, but not in two non-cytolytic Th cell lines7. These findings suggest that the serine esterase is involved in cytolytic activity and that a variety of effector cells share a common cytolytic mechanism. To explore the role of the serine esterase in this process, we have been studying additional properties of the enzyme in murine T cells. We show here that (1) it is a membrane-associated, disulphide-linked dimer, (2) it has trypsin-like properties but is not a general protease, (3) in density gradient centrifugation it sediments with perforin, (4) it is secreted by Tc cells during their cytolytic attack on target cells, and (5) antiserum to Tc-cell serine esterase reacts with the enzyme in Th/c cells.

Two antibody-secreting murine hybridomas, F1G3.1 and F2A11.5, have been established from B10.D2 mice immunized with cells from the murine cytotoxic T-lymphocyte clone G4. The two clones used, G4 and B10, were derived from BALB.B (H-2b) mice and the target antigen specificity of both maps to the Dd region of the murine H-2 complex. However, B10 has a lower affinity for the target cells, as shown by its lower specific killing of blasts and its higher susceptibility to blocking by anti-Lyt-2 monoclonal antibody 53-6.75. The monoclonal antibodies, F1G3.1 and F2A11.5, react only with cells from clone G4. Similarly, they block only the specific cytolysis mediated by G4; no effect on cytotoxicity mediated by B10 or by heterogeneous populations of cytotoxic T lymphocytes was found. F1G3.1, especially, is very active in stimulating G4 to secrete immune interferon; B10 in contrast did not show any induction on treatment with these monoclonal antibodies. The structure of the surface antigen on G4 cells recognized by these monoclonal antibodies was revealed by immunoprecipitation studies of radioiodinated cell surface proteins. A protein dimer could be identified with an apparent molecular size of 80,000 daltons consisting of monomers migrating as 42,000-dalton proteins on reduction. So far, electrophoresis in the presence of NaDodSO4 does not indicate any heterogeneity in the size of the monomers. This molecule can be distinguished from the Lyt-2 complex.

A small set of concanavalin A (Con A)-binding glycoproteins was isolated from the surface membrane of cloned cytotoxic T lymphocytes (CTL) and partly identified using monoclonal antibodies. The binding of Con A by these glycoproteins on the CTL surface results in the secretion of gamma-interferon and in blocking the effector functions of the cells-namely, antigen-specific and lectin-dependent cytotoxicity. The Con A is evidently bound tightly to some surface structures ("Con A-receptors") that are required for the activation and cytotoxic activity of CTL. To isolate and identify these receptors, antibodies to Con A were used. After Con A was allowed to bind to radiolabeled cloned CTL (labeled with 125I or [35S]methionine or 3H-labeled amino acids), the cells were washed thoroughly, lysed in detergents and anti-Con A antibodies were added to bind to the Con A-receptor complexes. The resulting aggregates were adsorbed with protein A-bearing Staphylococci and the receptors were then specifically released from the pelleted bacteria by alpha-methyl-D-mannoside and analyzed by polyacrylamide gel electrophoresis under reducing conditions. Eight to nine labeled components were seen by autoradiography and with the aid of monoclonal antibodies to known T-cell surface molecules, four were identified as T200, lymphocyte function-associated antigen (LFA)-1, alpha- and beta-chains, and (on some clones) Lyt-2. Other components with Mr congruent to 160,000, 120,000, 46,000, 42,000, and 23,000 have not been identified. The procedures described here may have general application in the studies of the functional properties of other cell surface molecules.

Interferon (IFN) was found to be secreted by cloned lines of murine cytotoxic T lymphocytes in response to mitogenic or antigen specific stimulation. The IFN produced was shown to be of the immune type based on its sensitivity to pH 2.0 and on the ability of an antiserum to immune IFN to neutralize the antiviral activity.

Cytotoxic T (Tc) lymphocytes recognize and lyse target cells and are thought to serve as an important defence against viral infections and possibly against neoplasms. The nature of the receptors responsible for antigen recognition by these cells is becoming clearer, but the molecular mechanisms responsible for their cytolytic activity remain largely unknown. The possibility that proteases are involved in this process has been suggested by the effects of certain inhibitors. Here we demonstrate that clones of murine Tc cells possess considerable trypsin-like esterase activity when assayed by a sensitive colorimetric assay. This activity was blocked completely by two serine esterase inhibitors, diisopropylfluorophosphoridate (DFP) and phenylmethylsulphonyl fluoride (PMSF), but not by N alpha-tosyl lysyl chloromethyl ketone (TLCK). The use of 3H-DFP as an affinity-labelling reagent demonstrated that the esterase activity resides in a protein of relative molecular mass (Mr) 28,000 (28K). A wide variety of other lymphocytes, including those from thymus, spleen and lymph node, established lines of B cells and noncytotoxic T cells, and clones of T helper cells, had about 300-fold less esterase activity than the Tc-cell clones and far smaller amounts of the DFP-reactive 28K protein. However, in thymocytes the esterase activity increased 20-50-fold and the 28K protein became more prominent 4 days after these cells had been stimulated in vitro to generate Tc cells.

Immune responses begin with the binding of antigen to surface receptors on lymphocytes. In this reaction, called ‘antigen recognition’, lymphocyte receptors discriminate by variations in the strength of the binding reaction among vast numbers of antigens, including many that differ from each other only very slightly in structure. Antigen recognition by B lymphocytes is now well understood: their recognition molecules are cell surface antibodies (Warner, 1974) and antigen binding by antibodies has been characterized, for many antigens, at a highly sophisticated chemical level. However, antigen recognition by T cells (i.e. thymus-derived lymphocytes) is poorly understood and remains one of the most pressing challenges of modern immunology.