Comparative lymphatic, ocular, and metabolic phenotypes of Foxc2 haploinsufficient and aP2-FOXC2 transgenic mice.

FOXC2 mutations cause the lymphatic/ocular disorder Lymphedema-Distichiasis (LD), and Foxc2 haploinsufficient mice mimic this disorder. To determine if FOXC2 overexpression might also cause lymphatic and/or ocular abnormalities, we performed dynamic lymphatic imaging (Evans blue dye), ocular tissue examination, and metabolic profiles in mice: transgenic for FOXC2 with an adipocyte (aP2) promoter (aP2-FOXC2 Tg), heterozygous for targeted disruption of Foxc2 (Foxc2+/-), or compound heterozygous and transgenic (Foxc2+/-, Tg) compared to wild-type controls (WT). Foxc2+/-; aP2-FOXC2 Tg; and Foxc2+/-, Tg, exhibited LD's distinctive hyperplastic lymphatic phenotype characterized by increased number of lymphatic channels and lymph nodes as well as retrograde lymph reflux. Foxc2+/-, and Foxc2+/-, Tg but not aP2-FOXC2 Tg or WT showed an abnormal ocular phenotype. Previously described alterations in brown/ white fat distribution and lean phenotype in aP2-FOXC2 transgenics were confirmed. AP2-FOXC2 Tg immunohistochemistry disclosed aberrant FOXC2 expression in ectopic sites, especially embryonic heart. Lymphatic system links with fat metabolism are discussed.

Fifth vaccination with dipthteria, tetanus and acellular pertussis is beneficial in four- to six-year-olds.

OBJECTIVE: Diphtheria, tetanus and pertussis serum antibody titers were assessed before a fifth dose of diphtheria-tetanus-acellular pertussis (DTaP) or diphtheria-tetanus-whole cell pertussis (DTwP) vaccination at age 4 to 6 years. METHODS: Healthy children who had participated in a series of National Institutes of Health-sponsored trials assessing DTwP and DTaP vaccines provided prevaccination sera before a fifth dose of DTwP or DTaP. The trial design was prospective, randomized and double blind. Diphtheria, tetanus and pertussis antibody titers were measured by enzyme-linked immunosorbent assay. Pertussis results are expressed in enzyme-linked immunosorbent assay units/ml based on US Food and Drug Administration reference sera. Tetanus and diphtheria toxin concentrations are expressed in IU/ml with a WHO international reference sera as a standard. RESULTS: For diphtheria 100% of the children had antibody titers above the minimum protective level of 0.01 IU/ml and 86 to 100% (depending on prior vaccine product) had titers >0.1 IU/ml. However, only 0 to 40% of the children had antibody titers > or =1.0 IU/ml, a titer associated with more certain durable protection. For tetanus none of the children had an antibody titer below 0.01 IU/ml, and 93 to 100% had titers > or =0.1 IU/ml, a titer associated with more certain, durable protection. For pertussis the geometric mean concentrations of antibody before booster were uniformly very low, and the percentage of children exceeding the minimum detectable titer of antibody by 4-fold was also low. CONCLUSION: Before a 4- to 6-year-old booster, a large proportion of children have titers of antibody to diphtheria below the certain, durable protective level. Because serologic correlates and minimum protective titers of antibody to pertussis antigens have not been established, the relevance of the low titers determined in the current study is unknown but a potential concern.

Safety and immunogenicity of four doses of Neisseria meningitidis group C vaccine conjugated to CRM197 in United States infants.

BACKGROUND: Following widespread use of conjugate pneumococcal vaccine, Neisseria meningitidis likely will become the leading cause of bacterial sepsis and meningitis in US children. This report describes the safety and immunogenicity in US children of four consecutive doses of a meningococcal group C vaccine conjugated to CRM197 via reductive amination (MnCC). METHODS: One hundred six healthy 2-month-old infants received MnCC at 2, 4 and 6 months of age in a randomized, controlled double blind study; children in the other treatment arm were given a 7-valent conjugate pneumococcal vaccine. Parents reenrolled 64 of these children at 12 to 15 months to receive a fourth dose of MnCC. Routine childhood vaccines, including DTP, were coadministered. Temperatures and symptoms were recorded for 3 days after each immunization. Serum enzyme-linked immunosorbent assay IgG and bactericidal antibodies were measured prevaccination and before and 1 month after Doses 3 and 4. RESULTS: Moderate to severe local reactions, defined as erythema or induration > or =2.4 cm or pain that interfered with limb movement was reported after 0 to 3.2% of MnCC injections, depending on the reaction and dose. Fever occurred in 23 to 37% of children, but the contribution of MnCC to the febrile reactions is unknown. Geometric mean concentrations of IgG antibody to group C meningococcal polysaccharide were 3.72 microg/ml after Dose 3 and 8.03 microg/ml after the booster. Geometric mean functional serum bactericidal antibody titers after Doses 3 and 4 were 1:463 and 1:2341, respectively. One hundred percent of children had a serum bactericidal antibody titer of > or =1:64 after three doses and > or = 1:128 after the booster. CONCLUSIONS: The MnCC vaccine had an acceptable safety profile and generated high titers of bactericidal antibody in immunized US infants and toddlers. It appears to be an attractive candidate vaccine for the prevention of serogroup C meningococcal disease in young children.

Diminution of the anti-polyribosylribitol phosphate response to a combined diphtheria-tetanus-acellular pertussis/Haemophilus influenzae type b vaccine by concurrent inactivated poliovirus vaccination.

BACKGROUND: Prelicensure studies of Haemophilus influenzae type b vaccines (Hib) and diphtheria-tetanus-acellular pertussis vaccines (DTaP) were evaluated with concurrent oral poliovirus vaccine (OPV). However, inactivated poliovirus vaccine (IPV) is now recommended. A trial was conducted in which infants received a DTaP and Hib vaccine, separately (+) or combined (/), with either all OPV, all IPV or sequential IPV-OPV for the primary series of vaccinations. METHODS: In this protocol 567 infants were equally randomized to receive one of the following: Reference Arm A, DTaP + Hib + OPV; Treatment Arm B, DTaP/Hib + OPV; Treatment Arm C, DTaP/Hib + IPV at 2 and 4 months and OPV at 6 months; or Treatment Arm D, DTaP/Hib + IPV. antibodies against all administered antigens were measured at 7 months of age. Children with an antibody response to Hib (anti-polyribosylribitol phosphate (anti-PRP) <0.15 microg/ml had an antibody titer repeated after the toddler booster immunization. RESULTS: A significant diminution in the anti-PRP response was observed at 7 months of age in children given two or three doses of IPV concurrently with DTaP/Hib, compared with the groups given OPV. The geometric mean concentration of anti-PRP, percentage of children with > or = 0.15 microg/ml and percentage of children with > or = 1.0 microg/ ml, respectively, were: A, 4.4, 98%, 81%; B, 3.2, 94%, 78%; C, 1.3, 86%, 58% and D, 1.2, 84%, 53%. CONCLUSION: In this trial concurrent IPV appeared to interfere with the anti-PRP response to DTaP/Hib vaccine, suggesting that introduction of new vaccines may require evaluation of immune responses to all concurrently administered vaccines.

Safety and immunogenicity of six acellular pertussis vaccines and one whole-cell pertussis vaccine given as a fifth dose in four- to six-year-old children.

OBJECTIVE: To evaluate the safety and immunogenicity of 6 different acellular pertussis vaccines combined with diphtheria and tetanus toxoids (DTaP) and with 1 licensed whole-cell pertussis vaccine (DTwP) as a fifth dose in children who had previously received the same DTaP, a different DTaP, or DTwP as primary and fourth-dose vaccinations. METHODS: Healthy 4- to 6-year-old children were enrolled at 5 National Institute of Allergy and Infectious Diseases Vaccine Treatment and Evaluation Units to receive a fifth dose of a DTaP or DTwP vaccine. All had been randomly assigned to receive 3 primary doses of DTaP or DTwP at 2, 4, and 6 months and a fourth-dose booster at 15 to 20 months of age as part of earlier National Institutes of Health multicenter acellular pertussis vaccine trials. Parents recorded the occurrence and magnitude of fever, irritability, and injection site redness, swelling, and pain for 3 days after vaccination. Sera obtained before and 1 month after the booster vaccination were analyzed by enzyme-linked immunosorbent assay for antibody to pertussis toxin, filamentous hemagglutinin, fimbriae, pertactin, and diphtheria and tetanus toxoid. Safety and/or immunogenicity data are reported for 317 children who received DTaP and 10 children who received DTwP. RESULTS: Fever and moderate or severe irritability were uncommon following the fifth dose of DTaP vaccine and were generally less frequent than following the fourth dose. However, for the DTaP vaccine groups, redness, swelling, and pain increased in prevalence compared with the fourth dose. The time course and frequency of reactions following DTaP vaccination were generally similar in children who received the same DTaP, a different DTaP, or DTwP for previous doses in the 5- dose series. No significant differences among the DTaP vaccines were detected in the occurrence of reactions, but the statistical power to detect differences was limited by sample size. Significant increases in antibodies directed against the included antigens were observed for all DTaP vaccines in paired pre- and post-fifth dose sera. Post-fifth dose antibody concentrations differed significantly among the DTaP vaccines. Some children in the study showed an antibody response to an antigen not reported to be in the DTaP vaccine. CONCLUSION: All the studied DTaP vaccines performed similarly with regard to reactions, whether given as a fifth sequential dose of the same vaccine, a mix of different DTaP vaccines in the 5-dose sequence, or after 3 DTwP and 1 DTaP vaccinations. Large injection site reactions occurred more frequently after the fifth dose of DTaP than after the previous 4 doses. A fifth dose of all DTaP vaccines induced an antibody response to those antigens contained in the vaccine. No DTaP was consistently most or least reactogenic or immunogenic.

Extensive swelling after booster doses of acellular pertussis-tetanus-diphtheria vaccines.

BACKGROUND: Diphtheria and tetanus toxoid combined with acellular pertussis (DTaP) vaccines are less reactogenic than diphtheria and tetanus toxoid combined with whole cell pertussis (DTwP) vaccines. However, local reactions increase in rate and severity with each successive DTaP dose, and swelling of the entire injected limb has been reported after booster doses. METHODS: We reviewed reports of swelling of the entire thigh or upper arm after the fourth and fifth dose, respectively, of DTaP vaccines administered in the National Institutes of Health multicenter comparative DTaP studies. Relationships were explored among reports of severe swelling, rates of other reactions, quantity of vaccine contents, and prevaccination and postvaccination antibody levels to pertussis toxin, tetanus toxin, and diphtheria toxin. RESULTS: Entire thigh swelling was an unsolicited reaction reported in 20 (2%) of the 1015 children who received 4 consecutive doses of the same DTaP vaccine. The reaction was associated with 9 of the 12 DTaP vaccines evaluated. Although there were no reports of swelling of the entire upper arm in 121 children given a fifth dose of the same DTaP, 4 (2.7%) of 146 recipients of 5 doses of a mixed schedule of DTaP vaccines experienced such swelling. Rates of other reactions were higher in children with entire thigh swelling than in those without. Of the children with entire thigh swelling, 60% had local pain, and 60% had erythema. All swelling subsided spontaneously without sequelae. There was a significant linear association between the rates of entire thigh swelling after dose 4 and diphtheria toxoid content in the DTaP products. Lesser degrees of swelling (>50 mm but less than entire limb) correlated with pertussis toxoid content after dose 4 and aluminum content after dose 5. No relationship was established between levels of serum antibody to diphtheria, tetanus, or pertussis toxin and rates of swelling of the whole thigh. CONCLUSIONS: Booster doses of DTaP vaccines can cause entire limb swelling, which is usually associated with redness and pain. Our data suggest that this extensive swelling reaction may be more common with vaccines containing high diphtheria toxoid content.

Pneumococcal vaccines.

Despite advances in our knowledge of the pathogenesis and host immune response and efforts at prevention and treatment, S. pneumoniae continues to cause considerable disease and mortality. The currently available polysaccharide vaccines confer moderate protection in most adults and older children, but they are underutilized. There are an estimated 40,000 deaths per year among U.S. adults due to pneumococcus, of which nearly half might be prevented if the current recommendations for vaccination were followed. Future advances must include augmented efforts at coverage of older children and adults via improvements in education, delivery mechanisms and financing. The major advance in the field is the development of conjugate vaccines which appear safe, immunogenic and efficacious against invasive pneumoccocal disease in infants. The first conjugate vaccine is anticipated to be available for use in the U.S. in the year 2000.

Oral tetravalent rotavirus vaccine can be successfully coadministered with oral poliovirus vaccine and a combined diphtheria, tetanus, pertussis and Haemophilus influenzae type b vaccine. US Rhesus Rotavirus Vaccine Study Group.

AIM: To determine whether an oral tetravalent rotavirus vaccine (RV-TV) can be safely coadministered with a combined diphtheria-tetanus-pertussis-Haemophilus influenzae type b vaccine (DTP/Hib) and oral poliovirus vaccine (OPV) to healthy infants without interfering with the immune responses to any of the component antigens. METHODS: Two hundred sixty-seven infants ages 2 to 3 months were randomly assigned in a double blind fashion to receive three doses of either placebo or RV-TV, each containing 4 x 10(5) plaque-forming units, concurrently with DTP/ Hib (Tetramune) and OPV at approximately 2, 4 and 6 months of age. Infants were followed for 5 days after each dose for the occurrence of adverse events and subsequently until 3 to 6 weeks after the third dose of RV-TV or placebo. Immune responses were assessed by measuring the postvaccination serum antibody titers to each component of DTP/ Hib and OPV at 3 to 6 weeks after the third dose. RESULTS: The percentage of infants who attained protective antibody titers and the distribution of antibody titers against diphtheria toxoid, tetanus toxoid and H. influenzae type b were not statistically different between RV-TV and placebo recipients. The distribution of antibody titers against different antigens of Bordetella pertussis (agglutinins, pertussis toxoid, filamentous hemagglutinin, fimbriae antigens and the 69-kDa outer membrane protein) was compared and no significant differences were found. The percentage of infants with detectable neutralizing antibodies against the three serotypes of poliovirus and the distribution of antibody titers was not statistically different between RV-TV and placebo recipients. There were no clinically meaningful differences in postvaccination reactions between RV-TV and placebo recipients. CONCLUSIONS: Three doses of RV-TV can be safely coadministered with three doses of DTP/ Hib and OPV without diminishing an infant's serum antibody responses to each component of these vaccines. Therefore RV-TV can be given at the standard childhood visits at 2, 4 and 6 months of age.

Safety and immunogenicity of heptavalent pneumococcal vaccine conjugated to CRM197 in United States infants.

OBJECTIVE: To determine the safety and immunogenicity of heptavalent pneumococcal saccharide vaccine (serotypes 4, 6B, 9V, 14, 18C, 19F, 23F) individually conjugated to CRM197 (PNCRM7), administered at 2, 4, 6, and 12 to 15 months of age. DESIGN: Two hundred twelve healthy 2-month-old infants were equally randomized to receive four consecutive doses of PNCRM7 or an investigational meningococcal group C conjugate vaccine, which served as a control. Concomitantly administered routine vaccines were oral polio vaccine and combined diphtheria toxoid, tetanus toxoid, and whole cell pertussis vaccine/Haemophilus influenzae type b vaccine consisting of capsular oligosaccharides conjugated to CRM197 (DTP/HbOC) at 2, 4, and 6 months, and either measles-mumps-rubella vaccine or HbOC at 12 to 15 months. Active safety surveillance was conducted for 3 days after each dose. Antibody concentrations to each of the 7 pneumococcal serotypes were measured by enzyme-linked immunosorbent assay prevaccination, after doses two and three, prebooster, and postbooster. RESULTS: Significantly fewer children experienced local reactions at the PNCRM7 injection site than at the DTP/HbOC site. There was no increase in the incidence or severity of local reactions at the PNCRM7 site with increasing doses of vaccine. Mild to moderate postvaccination fever was common in both the PNCRM7 and control vaccine groups, however DTP/HbOC was administered concurrently. All 7 vaccine serotypes were immunogenic. The kinetics of the immune responses were serotype-specific. After three doses of PNCRM7, between 92% to 100% of children had >/=0.15 microg/mL of antibody, and 51% to 90% achieved a level of >/=1 microg/mL against specific serotypes. A booster dose of PNCRM7 resulted in a brisk anamnestic response to all 7 vaccine serotypes, demonstrating effective stimulation of T-cell memory by the primary series of vaccinations. CONCLUSION: Primary immunization followed by a booster dose of PNCRM7 seemed to be acceptably safe and resulted in significant rises in antibody to all 7 serotypes. Implications. Studies to assess vaccine efficacy of PNCRM7 for prevention of systemic disease, nasopharyngeal colonization, and acute otitis media are in progress. If PNCRM7 proves to be protective, there is the potential to prevent up to 85% of invasive pneumococcal disease occurring in US children.

A safety and immunogenicity comparison of 12 acellular pertussis vaccines and one whole-cell pertussis vaccine given as a fourth dose in 15- to 20-month-old children.

OBJECTIVE: To compare the safety and immunogenicity of 12 different acellular pertussis vaccines combined with diphtheria and tetanus toxoids (DTaP) with one licensed diphtheria, tetanus, and whole-cell pertussis vaccine (DTwP) as a fourth-dose booster in children who had previously received DTaP or DTwP primary vaccinations. METHODS: Healthy 15- to 20-month-old children were enrolled at six National Institutes of Health Vaccine Treatment and Evaluation Units. All had been randomly assigned to receive three primary doses of DTaP or DTwP at 2, 4, and 6 months of age as part of an earlier National Institutes of Health multicenter trial of DTaP vaccines in the same Vaccine Treatment and Evaluation Units. Parents recorded the occurrence and magnitude of fever; irritability; and injection site redness, swelling, and pain for 3 days after vaccination. Sera obtained before and 1 month after the booster vaccination were analyzed for antibody to pertussis toxin (PT), filamentous hemagglutinin (FHA), fimbriae (FIM), and pertactin (PRN). Diphtheria and tetanus toxoid as well as PT neutralizing (Chinese hamster ovary cell) and whole-cell agglutinating antibodies were measured on a subset of sera. RESULTS: A total of 1293 children contributed fourth-dose reaction data. Reactions were less frequent after DTaP than after DTwP. For children vaccinated with a fourth dose of DTaP, which was the same DTaP as received in the primary series, fever and injection site redness, swelling, and pain increased in prevalence compared with the third dose in the primary series. For children receiving DTaP as a fourth dose, injection site redness and swelling occurred more frequently in DTaP-primed than in DTwP-primed children. Variation in the occurrence of reactions among DTaP vaccines was observed. A total of 1160 paired pre- and postvaccination sera were available for analysis. Serum antibody concentrations before boosting were lower than those obtained 1 month after the primary immunization. After the fourth dose, significant increases in antibodies directed against the included antigens were observed for all vaccines; postbooster vaccination antibody titers differed significantly among the DTaP vaccines. For children primed and boosted with the same DTaP, antibody levels were not directly related to the quantity of antigen included for PT, FHA, and FIM; for PRN, there was a closer relationship. Some DTaP vaccines given as fourth-dose boosters elicited antibody to PRN or FIM in some vaccinees, although the DTaP vaccines were not reported to contain these antigens; these responses were observed more frequently in DTwP-primed children. Agglutinin antibody rises were observed in all groups immunized with four doses of a DTaP vaccine containing FHA or PRN, regardless of whether the vaccine included FIM. Diphtheria and tetanus antibody levels exceeded the presumed protective concentration (0.1 IU/mL for diphtheria and 0.01 IU/mL for tetanus) after the fourth dose for all vaccinees. CONCLUSION: Although differences were observed in reaction rates among the DTaP vaccines given as a fourth dose, the DTaP vaccines were, in general, associated with fewer adverse events than a US-licensed DTwP. For DTaP vaccines, fever; irritability; and injection site pain, redness, and swelling occurred more frequently after the fourth dose than after the third dose of the same vaccine in the primary series. No DTaP was consistently most or least reactogenic or immunogenic. Although serologic correlates of pertussis immunity are not defined, it is clear that most DTaP vaccines can stimulate comparable or higher serum antibody responses than DTwP for those antigens contained in the vaccine.

Infant immunization with pneumococcal CRM197 vaccines: effect of saccharide size on immunogenicity and interactions with simultaneously administered vaccines.

Six pentavalent pneumococcal conjugate vaccines (Pn-CRM197) were evaluated among 400 infants. The vaccines differed in saccharide chain length (oligosaccharide [OS] or polysaccharide [PS]) and saccharide quantity (0.5, 2, or 5 microg). Subjects were randomized into groups 1-6 (Pn-CRM197 recipients) or 7 (controls) for immunization at 2, 4, and 6 months of age. Pn-CRM197 were well tolerated and elicited mean antibody concentrations that exceeded those in controls for all 5 capsular serotypes. PS formulations were generally more immunogenic than their OS counterparts. For PS vaccines, a dose-response was documented (5 microg > 2 microg > 0.5 microg), but the differences between the 5- and 2-microg formulations were insignificant. The mean anti-PRP antibody concentration was significantly higher among Pn-CRM197 recipients. It is concluded that PS vaccines are more immunogenic than OS vaccines. The improved immunogenicity from Haemophilus type b oligosaccharide conjugate (HbOC) vaccine when given with Pn-CRM197 suggests that a decreased dose of HbOC vaccine may be sufficient to elicit protection.

Influence of breast-feeding and oral poliovirus vaccine on the immunogenicity and efficacy of rotavirus vaccines.

To be practical and cost effective, rotavirus vaccines will have to be administered to infants with routine childhood vaccinations. Rotavirus vaccine studies were reviewed to evaluate the effect of breast-feeding and concurrent oral poliovirus vaccination (OPV) on the immunogenicity and efficacy of oral rotavirus vaccines. Both breast-feeding and OPV appeared to interfere, to some degree, with the serum immune response to rotavirus vaccines; however, the effects were usually not statistically significant and were largely overcome by administering three doses of rotavirus vaccine rather than one. Available data show no decrease in the protective efficacy of rotavirus vaccine in breast-fed children or in children receiving concurrent OPV, but larger sample sizes will be required to definitely rule out any effect.

Concurrent oral poliovirus and rhesus-human reassortant rotavirus vaccination: effects on immune responses to both vaccines and on efficacy of rotavirus vaccines. The US Rotavirus Vaccine Efficacy Group.

Interference between oral poliovirus vaccine (OPV) and monovalent (RRV-S1) and tetravalent (RRV-TV) rhesus-human rotavirus vaccines was evaluated. Serum antibody responses to OPV and rotavirus vaccines and efficacy of rotavirus vaccines were compared among control and vaccine groups stratified by number of concurrent OPV and rotavirus vaccinations received. Neutralizing antibody titers to poliovirus type 1 tended to rise more steeply in placebo than RRV-TV recipients, but there were no significant differences in seroprevalence or in geometric mean titers (GMTs) of antibodies to types 1, 2, or 3 among groups. Concurrent OPV resulted in lower IgA GMTs to rotavirus in RRV-S1 but not RRV-TV recipients. Rotavirus gastroenteritis rates among rotavirus vaccines did not differ by number of concurrent OPV doses received, but the sample sizes were too small to rule out any effect. These results suggest OPV and rhesus-human rotavirus vaccines may be given at the same visit in the United States.

Safety and efficacy of high-dose rhesus-human reassortant rotavirus vaccines--report of the National Multicenter Trial. United States Rotavirus Vaccine Efficacy Group.

OBJECTIVE: Rotavirus is a leading cause of morbidity and mortality from dehydrating gastroenteritis in infants and young children worldwide. Virtually every child is infected by age 4 years, justifying universal childhood immunization when a safe and effective vaccine is available. We report the results of a multicenter, placebo-controlled field trial in the United States of monovalent serotype 1 and tetravalent (TV) rhesus-human reassortant rotavirus vaccines (RRVs). DESIGN: In this randomized, double-blind trial, 1278 healthy infants ages 5 to 25 weeks received three oral doses of RRV serotype 1, RRV-TV, or a placebo at approximately 2, 4, and 6 months of age. Vaccines contained 4 x 10(5) plaque-forming units of virus. Gastroenteritis episodes were monitored, and severity was graded throughout one rotavirus season. Two stool specimens per episode were tested for rotavirus. RESULTS: The incidence of reactions did not differ among treatment groups during the 5-day, postvaccination safety surveillance period for any of the three doses. Both vaccines significantly reduced the incidence of rotavirus gastroenteritis. Vaccination was most protective against serious rotavirus illness; RRV-TV prevented 49% of rotavirus episodes, 80% of very severe episodes, and 100% of dehydrating rotavirus illness. Reduction of rotavirus disease by RRV-TV resulted in significantly fewer total episodes of gastroenteritis of all causes and an 82% reduction in all cases of dehydrating diarrhea. CONCLUSION: RRV-TV is highly protective against very severe, dehydrating rotavirus gastroenteritis.