The heptavalent pneumococcal-CRM197 conjugate vaccine (PCV-7) has been incompletely studied in very-low-birth-weight (VLBW, ≤1500 grams) infants.
To assess PCV-7 immunogenicity in VLBW, premature infants. We hypothesized that the frequency of post-vaccine antibody concentrations ≥0.15 µg/mL would vary directly with birth weight.
This was a multi-center observational study. Infants 401–1500 grams birth weight and <32 0/7 weeks gestation, stratified by birth weight, were enrolled from 9 NICHD Neonatal Research Network centers. Infants received PCV-7 at 2, 4 and 6 months after birth and had blood drawn 4–6 weeks following the third dose. Antibodies against the 7 vaccine serotypes were measured by enzyme-linked immunosorbent assay.
Of 369 enrolled infants, 244 completed their primary vaccine series by 8 months and had serum obtained. Subjects were 27.8 ± 2.2 (mean ± standard deviation) weeks gestation and 1008 ± 282 grams birth weight. Twenty-six percent had bronchopulmonary dysplasia and 16% had received postnatal glucocorticoids. Infants 1001–1500 grams birth weight were more likely than those 401–1000 grams to achieve antibody concentrations ≥0.15 µg/mL against the least two immunogenic serotypes (6B: 96% v. 85%, P = 0.003 and 23F: 97% v. 88%, P = 0.009). In multiple logistic regression analysis, lower birth weight, postnatal glucocorticoid use, lower weight at blood draw and Caucasian race were each independently associated with antibody concentrations <0.35 µg/mL against serotypes 6B and/or 23F.
When compared with larger premature infants, infants weighing ≤1000 grams at birth have similar antibody responses to most, but not all, PCV-7 vaccine serotypes.
Infant, premature; infant, very low birth weight; pneumococcal vaccines; immunization; vaccines
The safety and effectiveness of meropenem in young infants with suspected or confirmed intra-abdominal infections were evaluated. was well tolerated in this cohort of critically-ill infants, and the majority of infants treated with meropenem (84%) met the definition of therapeutic success.
Background. Intra-abdominal infections are common in young infants and lead to significant morbidity and mortality. Meropenem is a broad-spectrum antimicrobial with excellent activity against pathogens associated with intra-abdominal infections. The purpose of this study was to determine the safety and effectiveness of meropenem in young infants with suspected or complicated intra-abdominal infections.
Methods. Preterm and term infants <91 days of age with suspected or confirmed intra-abdominal infections hospitalized in 24 neonatal intensive care units were studied in an open-label, multiple-dose study. Adverse events and serious adverse events were collected through 3 and 30 days following the last meropenem dose, respectively. Effectiveness was assessed by 3 criteria: death, bacterial cultures, and presumptive clinical cure score.
Results. Of 200 subjects enrolled in the study, 99 (50%) experienced an adverse event, and 34 (17%) had serious adverse events; no adverse events were probably or definitely related to meropenem. The most commonly reported adverse events were sepsis (6%), seizures (5%), elevated conjugated bilirubin (5%), and hypokalemia (5%). Only 2 of the serious adverse events were determined to be possibly related to meropenem (isolated ileal perforation and an episode of fungal sepsis). Effectiveness was evaluable in 192 (96%) subjects, and overall treatment success was 84%.
Conclusions. Meropenem was well tolerated in this cohort of critically ill infants, and the majority of infants treated with meropenem met the definition of therapeutic success.
Clinical Trials Registration. NCT00621192.
To assess the extent and nature of somatic categorical selection of CDR-H3 content in peritoneal cavity (PerC) B cells, we analyzed the composition of VH7183DJCμ transcripts derived from sorted PerC B-1a, B-1b, and B-2 cells. We divided these sequences into those that contained N nucleotides (N+) and those that did not (N−), and then compared them to sequences cloned from sorted IgM+IgD+ B cells from neonatal liver and both wild-type and TdT deficient adult bone marrow. We found that the PerC B-1a N− repertoire is enriched for the signatures of CDR-H3 sequences present in neonatal liver and shares many features with the B-1b N− repertoire, whereas the PerC B-1a N+, B-1b N+ and B-2 N+ repertoires are enriched for adult bone marrow sequence signatures. However, we also found several sequence signatures that were not shared with other mature perinatal or adult B cell subsets; but were either unique or variably shared between the two or even among all three of the PerC subsets that we examined. These signatures included an increased number of sequences lacking N nucleotides in the B-2 population and an increased use of DH reading frame 2 which created CDR-H3s of greater average hydrophobicity. These findings provide support for both ontogenetic origin and shared antigen receptor-influenced selection as the mechanisms that shape the unique composition of the B-1a, B-1b and B-2 repertoires. The peritoneal cavity may thus serve as a general reservoir for B cells with antigen binding specificities that are uncommon in other mature compartments.
B cells; Antibodies; Gene Rearrangement; Repertoire Development
Information on cytokine profiles in fungal sepsis (FS), an important cause of mortality in extremely low birthweight infants (ELBW), is lacking. We hypothesized that cytokine profiles in the 1st 21 days of life in ELBW with FS differ from those with bacterial sepsis (BS) or no sepsis (NS).
In a secondary analyses of the NICHD Cytokine study, three groups were defined - FS (≥1 episode of FS), BS (≥1 episode of BS without FS), and NS. Association between 11 cytokines assayed in dried blood spots obtained on days 0-1, 3±1, 7±2, 14±3, and 21±3 and sepsis group was explored.
Of 1066 infants, 89 had FS and 368 had BS. Compared to BS, FS was more likely to be associated with lower birthweight, vaginal delivery, patent ductus arteriosus, postnatal steroids, multiple central lines, longer respiratory support and hospital stay, and higher mortality (p<0.05). Analyses controlling for covariates showed significant group differences over time for IFN-γ, IL-10, IL-18, TGF-β and TNF-α (p<0.05).
Significant differences in profiles for IFN-γ, IL-10, IL-18, TGF-β and TNF-α in FS, BS or NS in this hypothesis-generating secondary study require validation in rigorously designed prospective studies and may have implications for diagnosis and treatment.
To test the hypothesis that heart rate characteristics (HRC) monitoring improves neonatal outcomes.
Two-group, parallel, individually randomized controlled clinical trial of 3003 very low birth weight infants in 9 NICUs. In one group, HRC monitoring was displayed; in the other, it was masked. The primary outcome was number of days alive and ventilator-free in the 120 days after randomization. Secondary outcomes were mortality, number of ventilator days, NICU stay and antibiotic use.
Mortality was reduced in infants whose HRC monitoring was displayed, from 10.2% to 8.1% (HR = 0.78, 95% CI = 0.61 to 0.99, P = 0.04, number needed to monitor 48), and there was a trend toward increased days alive and ventilator-free (95.9 of 120 days compared to 93.6 in controls, P = 0.08). Mortality benefit was concentrated in infants with birth weight <1000g (HR=0.74, 95% CI 0.57 to 0.95, P=0.02, number needed to monitor 23). There were no significant differences in the other outcomes.
Heart rate characteristics monitoring can reduce mortality in very low birth weight infants.
Neonatal sepsis; sample entropy; predictive monitoring; heart rate variability
Suspected or complicated intra-abdominal infections are common in young infants and lead to significant morbidity and mortality. Meropenem is a broad-spectrum antimicrobial agent with excellent activity against pathogens associated with intra-abdominal infections in this population. The purpose of this study was to determine the pharmacokinetics (PK) of meropenem in young infants as a basis for optimizing dosing and minimizing adverse events.
Premature and term infants <91 days of age hospitalized in 24 neonatal intensive care units were studied. Limited PK sampling was performed following single and multiple doses of meropenem 20–30 mg/kg of body weight every 8–12 hours based on postnatal and gestational age at birth. Population and individual patient (Bayesian) PK parameters were estimated using NONMEM®.
Two hundred infants were enrolled and received study drug. One hundred eighty-eight infants with 780 plasma meropenem concentrations were analyzed. Their median (range) gestational age at birth and postnatal age at PK evaluation were 28 (23–40) weeks and 21 (1–92) days, respectively. In the final PK model, meropenem clearance (CL) was strongly associated with serum creatinine (SCR) and postmenstrual age (PMA) (CL [L/h/kg] = 0.12*[(0.5/SCR)**0.27]*[(PMA/32.7)**1.46]). Meropenem concentrations remained >4 μg/mL for 50% of the dose interval and >2 μg/mL for 75% of the dose interval in 96% and 92% of patients, respectively. The estimated penetration of meropenem into the cerebrospinal fluid was 70% (5–148).
Meropenem dosing strategies based on postnatal and gestational age achieved therapeutic drug exposure in almost all infants.
enterocolitis; necrotizing; infant; premature; cerebrospinal fluid
Cytokines mediate the host immune response to infectious microorganisms. The objective of this study was to determine whether immune regulatory interleukins (IL-4, IL-5, IL-6 and IL-10) and inflammatory cytokines (Interferon- [INF- ], tumor necrosis factor- [TNF- ], IL-2, and IL-17) are associated with an increased risk of developing blood stream bacterial/fungal infection (BSI) in extremely low birth weight (ELBW) infants. ELBW infants from 17 NICHD Neonatal Research Network centers without early onset sepsis were studied. Cytokines were measured from blood on days 1, 3, 7, 14, and 21 after birth. 996 ELBW infants contributed a minimum of 4080 unique measurements for each cytokine during the 5 sampling periods. Infants with BSI had lower levels of the inflammatory cytokines IL-17 (P=0.01), and higher levels of the regulatory cytokines, IL-6 (P=0.01) and IL-10 (P<0.001). Higher levels of regulatory cytokines relative to pro-inflammatory cytokines were associated with increased risk of BSI even after adjusting for confounding variables. In ELBW infants, the ratio of immune regulatory cytokines to inflammatory cytokines was associated with development of BSI. Altered maturation of regulatory and inflammatory cytokines may increase the risk of serious infection in this population.
The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT−/−) and wild-type (TdT+/+) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT−/− cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP19CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT−/− bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgMa and congenic TdT-sufficient CB17 IgMb bone marrow were placed in competition. TdT−/− cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.
Electronic supplementary material
The online version of this article (doi:10.1007/s00251-011-0543-7) contains supplementary material, which is available to authorized users.
Terminal deoxynucleotidyl transferase; B cell development; Immune responses; Mice
In mouse and human, the regulated development of antibody repertoire diversity during ontogeny proceeds in parallel with the development of the ability to generate antibodies to an array of specific antigens. Compared to adult, the human fetal antibody repertoire limits N addition and uses specifically positioned VDJ gene segments more frequently, including V6-1 the most DH-proximal VH, DQ52, the most JH-proximal DH, and JH2, which is DH-proximal. The murine fetal antibody repertoire also limits the incorporation of N nucleotides and uses its most DH proximal VH, VH81X, more frequently. To test whether DH and JH also follow the pattern observed in human, we used the scheme of Hardy to sort B lineage cells from BALB/c fetal and neonatal liver, RT-PCR cloned and sequenced VH7183-containing VDJCμ transcripts, and then assessed VH7183-DH-JH and complementary determining region 3 of the immunoglobulin heavy chain (CDR-H3) content in comparison to the previously studied adult BALB/c mouse repertoire. Due to the deficiency in N nucleotide addition, perinatal CDR-H3s manifested a distinct pattern of amino acid usage and predicted loop structures. As in the case of adult bone marrow, we observed a focusing of CDR-H3 length and CDR-H3 loop hydrophobicity, especially in the transition from the early to late pre-B cell stage, a developmental checkpoint associated with expression of the pre-B cell receptor. However, fetal liver usage of JH-proximal DHQ52 and DH-proximal JH2 was markedly greater than that of adult bone marrow. Thus, the early pattern of DH and JH usage in mouse feta liver mirrors that of human.
Electronic supplementary material
The online version of this article (doi:10.1007/s00251-010-0469-5) contains supplementary material, which is available to authorized users.
Fetal mouse repertoire; CDR-H3; Adult mouse repertoire
Extracorporeal membrane oxygenation (ECMO) is a life-saving support system used in neonates and young children with severe cardiorespiratory failure. Although ECMO has reduced mortality in these critically-ill patients, almost all patients treated with ECMO develop a systemic inflammatory response syndrome (SIRS) characterized by a ‘cytokine storm’, leukocyte activation, and multisystem organ dysfunction. We used a neonatal porcine model of ECMO to investigate whether rising plasma concentrations of inflammatory cytokines during ECMO reflect de novo synthesis of these mediators in inflamed tissues, and therefore, can be used to assess the severity of ECMO-related SIRS.
Three-week-old previously-healthy piglets were subjected to venoarterial ECMO for up to 8 hours. SIRS was assessed by histopathological analysis, measurement of neutrophil activation (flow cytometry), plasma cytokine concentrations (enzyme immunoassays), and tissue expression of inflammatory genes (polymerase chain reaction/western blots). Mast cell degranulation was investigated by measurement of plasma tryptase activity.
Porcine neonatal ECMO was associated with systemic inflammatory changes similar to those seen in human neonates. TNF-α and interleukin-8 (IL-8) concentrations rose rapidly during the first 2 hours of ECMO, faster than the tissue expression of these cytokines. ECMO was associated with increased plasma mast cell tryptase activity, indicating that increased plasma concentrations of inflammatory cytokines during ECMO may result from mast cell degranulation and associated release of preformed cytokines stored in mast cells.
TNF-α and IL-8 concentrations rose faster in plasma than in the peripheral tissues during ECMO, indicating that rising plasma levels of these cytokines immediately following the initiation of ECMO may not reflect increasing tissue synthesis of these cytokines. Mobilization of preformed cellular stores of inflammatory cytokines such as in mucosal mast cells may play an important pathophysiological role in ECMO-related SIRS.
ECMO; venoarterial; neonate; SIRS; mast cells; intestine; CRP; cytokines
In jawed vertebrates most expressed immunoglobulin H chains use only one of six possible DH reading frames. Reading frame 1 (RF1), the preferred reading frame, tends to encode tyrosine and glycine, whereas the other five RFs tend to be enriched for either hydrophobic or charged amino acids. Mechanisms proposed to favor use of RF1 include a preference for deletion over inversion that discourages use of inverted RF1, RF2 and RF3; sequence homology between the 5’ terminus of the JH and the 3’ terminus of the DH that promotes rearrangement into RF1; an ATG start site upstream of RF2 that permits production of a truncated Dμ protein; stop codons in RF3; and, following surface expression of IgM, somatic, presumably antigen receptor-based selection favoring B cells expressing Igs with tyrosine and glycine enriched CDR-H3s. By creating an IgH allele limited to the use of a single, frameshifted DFL16.1 DH gene segment, we tested the relative contribution of these mechanisms in determining reading frame preference. Dμ-mediated suppression via an allelic exclusion-like mechanism dominated over somatic selection in determining the composition of the CDR-H3 repertoire. Evidence of somatic selection for RF1-encoded tyrosine in CDR-H3 was observed, but only among the minority of recirculating, mature B cells which use DH in RF1. These observations underscore the extent to which the sequence of the DH acts to delimit the diversity of the antibody repertoire.
All jawed vertebrates limit use of DH reading frames (RFs) that are enriched for hydrophobic amino acids. In BALB/c mice, DFL16.1 RF2 encodes valine and isoleucine. To test whether increased use of RF2 affects B cell function, we examined B cell development and antibody production in mice with an IgH allele (ΔD-DµFS) limited to use of a single, frameshifted DFL61.1 gene segment. We compared the results of these studies to wild type mice, as well as those previously obtained in mice limited to use of either a single, normal DH or a single inverted DH that forces use of arginine in CDR-H3. All three of the mouse strains limited to a single DH produced fewer immature B cells than wild type. However, while mice limited to a single, normal DH achieved normal B cell numbers in the periphery, mice forced to preferentially use RF2 had reduced numbers of mature B cells in the spleen and bone marrow, mirroring the pattern previously observed in mice enriched for charged CDR-H3s. There were two exceptions. The mice using RF2 normally populated the marginal zone and peritoneal cavity whereas mice using inverted RF1 had increased numbers of marginal zone B cells and decreased numbers of B1a cells. When challenged with several T-dependent or T-independent antigens, antigen-specific antibody titers in the mice forced to use RF2 were altered. These findings indicate that B cell development and antigen specific antibody production can be heavily influenced by the global amino acid content of the CDR-H3 repertoire.
The power of the adaptive immune system to identify novel antigens depends on the ability of lymphocytes to create antigen receptors with diverse antigen-binding sites. For immunoglobulins, CDR-H3 lies at the center of the antigen binding site where it often plays a key role in antigen binding. It is created de novo by VDJ rearrangement and is thus the focus for rearrangement-dependent diversity. CDR-H3 is biased for the inclusion of tyrosine. In seeking to identify the mechanisms controlling CDR-H3 amino acid content, we observed that the coding sequence of DH gene segments demonstrate conservation of reading frame-specific sequence motifs, with RF1 enriched for tyrosine and depleted of hydrophobic and charged amino acids. Use of DH RF1 in functional VDJ transcripts is preferred from the earliest stages of B cell development, ‘pushing’ CDR-H3 to include specific categories of tyrosine enriched antigen binding sites. With development and maturation, the composition of the CDR-H3 repertoire appears to be ‘pulled’ into a more refined specific range. Forcing the use of alternative DH reading frames by means of gene targeting alters the expressed repertoire, enriching alternative sequence categories. This change in the repertoire variably affects antibody production and the development of specific B cell subsets.
Immunoglobulin; Diversity Gene Segment; Antibody Repertoire; B cell Development
Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3), the center of the classic antigen-binding site. To assess the role of DH RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single DH encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in DH RF1 alters CDR-H3 content and impairs B cell development and antibody production.
Nosocomial or late-onset sepsis is a common complication among premature infants, with a frequency inversely correlated with birth weight. Increased susceptibility to infection is due in part to an immature humoral (antibody-mediated) immune response. This study investigated the pharmacokinetics (PKs) and safety of a donor-selected specific intravenous immune globulin (IVIG) preparation, INH-A21 (Veronate), for prevention of sepsis in premature infants. Thirty-six infants weighing between 500 and 1,250 g during the first postnatal week were eligible to begin a series of up to four intravenous infusions of 500 or 750 mg/kg of body weight INH-A21. Blood samples were analyzed for antibodies against the Ser-Asp dipeptide repeat G (SdrG) and clumping factor A (ClfA) surface proteins of staphylococci. Sparse sampling and population PK analyses were performed to derive PK parameters. Following administration of the 500- and 750-mg/kg doses, the estimated average steady-state levels of anti-ClfA were 6.1 U/ml and 9.2 U/ml, respectively, and those of anti-SdrG were 5.2 U/ml and 7.7 U/ml, respectively. The elimination half-lives for anti-ClfA and anti-SdrG were 719 h and 701 h, respectively, and the clearances were 0.18 ml/h and 0.21 ml/h, respectively. In the final model, the values of the PK parameters were independent of gestational age. Both doses of INH-A21 were well tolerated, and the safety profile was similar to those of other IVIG preparations. These results suggest that a shorter dosing interval should be utilized between the first and second doses to achieve and maintain higher titers of anti-ClfA and anti-SdrG antibodies. Further studies examining INH-A21 for the prevention of late-onset sepsis in infants within the weight range studied are warranted.
The genital mycoplasmas represent a complex and unique group of microorganisms that have been associated with a wide array of infectious diseases in adults and infants. The lack of conclusive knowledge regarding the pathogenic potential of Mycoplasma and Ureaplasma spp. in many conditions is due to a general unfamiliarity of physicians and microbiology laboratories with their fastidious growth requirements, leading to difficulty in their detection; their high prevalence in healthy persons; the poor design of research studies attempting to base association with disease on the mere presence of the organisms in the lower urogenital tract; the failure to consider multifactorial aspects of diseases; and considering these genital mycoplasmas only as a last resort. The situation is now changing because of a greater appreciation of the genital mycoplasmas as perinatal pathogens and improvements in laboratory detection, particularly with regard to the development of powerful molecular nucleic acid amplification tests. This review summarizes the epidemiology of genital mycoplasmas as causes of neonatal infections and premature birth; evidence linking ureaplasmas with bronchopulmonary dysplasia; recent changes in the taxonomy of the genus Ureaplasma; the neonatal host response to mycoplasma and ureaplasma infections; advances in laboratory detection, including molecular methods; and therapeutic considerations for treatment of systemic diseases.
A PCR assay was used to analyze endotracheal aspirates from preterm infants for Ureaplasma parvum versus U. urealyticum. U. parvum was detected more often than U. urealyticum. There was no significant difference or trend in the prevalence of either species between infants with or without bronchopulmonary dysplasia when isolated alone.
Bacillus cereus is an uncommon but potentially serious bacterial pathogen causing infections of the bloodstream, lungs, and central nervous system of preterm neonates. A case of bacteremia caused by B. cereus in a 19-day-old preterm neonate who was successfully treated with vancomycin, tobramycin, meropenem, and clindamycin is described. Implications for the diagnostic laboratory and clinicians when Bacillus species are detected in normally sterile sites are discussed, and the small numbers of infant infections proven to be due to this organism that have been described previously are reviewed.