Prenatal Findings in Postnatal Cases of Disorders of Sex Development: Experience from a Tertiary-Specialized Center in Brazil.

INTRODUCTION AND OBJECTIVE: Prenatal suspicion of disorders/differences of sex development (DSDs) is a relatively new phenomenon. The aim of this study was to review the prenatal findings of DSD cases postnatally diagnosed in our tertiary referral center. METHODS: We evaluated 57 DSD cases with sex ambiguity who had undergone prenatal ultrasound with phenotypic sex assessment and/or cell-free fetal DNA (cffDNA) for genotypic sex assessment. RESULTS: Prenatal cffDNA had been performed in 32 cases, being positive (suggestive of male genotypic sex) in 26 and negative (suggestive of female genotypic sex) in 6. Five with cffDNA negative had a prenatal ultrasound indicating female external genitalia, in turn, in those with cffDNA positive, only two had a prenatal ultrasound indicating male external genitalia. Our postnatal data showed that when external genitalia were female or poorly virilized, prenatal ultrasound indicated female sex, but in cases of higher degree of virilization, ultrasound showed similar rates of male, female, or undetermined sex. Regarding the karyotype, our data showed those with XY karyotype had positive cffDNA, those with XX karyotype had negative cffDNA, and all five with sex chromosome anomalies had positive cffDNA because they were 45,X/46,XY. We suggested an algorithm to investigate these cases during gestation, including evaluation of uterus, fetal growth, and malformations. CONCLUSION: We suggest that the parents should be counseled prenatally by a dedicated multidisciplinary team with experience in DSD management and evaluated as soon as possible after birth.

DHX37 and NR5A1 Variants Identified in Patients with 46,XY Partial Gonadal Dysgenesis.

The group of disorders known as 46,XY gonadal dysgenesis (GD) is characterized by anomalies in testis determination, including complete and partial GD (PGD) and testicular regression syndrome (TRS). Several genes are known to be involved in sex development pathways, however approximately 50% of all cases remain elusive. Recent studies have identified variants in DHX37, a gene encoding a putative RNA helicase essential in ribosome biogenesis and previously associated with neurodevelopmental disorders, as a cause of PGD and TRS. To investigate the potential role of DHX37 in disorders of sexual development (DSD), 25 individuals with 46,XY DSD were analyzed and putative pathogenic variants were found in four of them. WES analyses were performed on these patients. In DHX37, the variant p.(Arg308Gln), recurrent associated with DSD, was identified in one patient; the p.(Leu467Val), predicted to be deleterious, was found together with an NR5A1 loss-of-function variant in patient 2; and, the p.(Val999Met) was identified in two unrelated patients, one of whom (patient 3) also carried a pathogenic NR5A1 variant. For both patients carrying DHX37 and NR5A1 pathogenic variants, a digenic inheritance is suggested. Our findings support the importance of DHX37 variants as a cause of disorders of sex development, implying a role in testis development.

Identification of single nucleotide variants in SLC26A9 gene in patients with cystic fibrosis (p.Phe508del homozygous) and its association to Orkambi® (Lumacaftor and Ivacaftor) response in vitro.

BACKGROUND: Since patients with cystic fibrosis with different Cystic Fibrosis Transmembrane Regulator (CFTR) genotypes present a wide response variability for modulator drugs such as Orkambi®, it is important to screen variants in candidate genes with an impact on precision and personalized medicine, such as Solute Carrier Family 26, member 9 (SLC26A9) gene. METHODS: Sanger sequencing for the exons and intron-exon boundary junctions of the SLC26A9 gene was employed in nine individuals with p.Phe508del homozygous genotype for the CFTR gene who were not under CFTR modulators therapy. The sequencing variants were evaluated by in silico prediction tools. The CFTR function was measured by cAMP-stimulated current (ΔIsc-eq-FSK) in polarized CFTR of human nasal epithelial cells cultured in micro-Ussing chambers with Orkambi®. RESULTS: We found 24 intronic variants, three in the coding region (missense variants - rs74146719 and rs16856462 and synonymous - rs33943971), and three in the three prime untranslated region (3' UTR) region in the SLC26A9 gene. Twenty variants were considered benign according to American College of Medical Genetics and Genomics guidelines, and ten were classified as uncertain significance. Although some variants had deleterious predictions or possible alterations in splicing, the majority of predictions were benign or neutral. When we analyzed the ΔIsc-eq-FSK response to Orkambi®, there were no significant differences within the genotypes and alleles for all 30 variants in the SLC26A9 gene. CONCLUSIONS: Among the nine individuals with p.Phe508del homozygous genotype for the CFTR gene, no pathogenic SLC26A9 variants were found, and we did not detect associations from the 30 SLC26A9 variants and the response to the Orkambi® in vitro.

SOX3 duplication in a boy with 46,XX ovotesticular disorder of sex development and his 46,XX sister with atypical genitalia: Probable germline mosaicism.

Ovotesticular disorders of sex development (OT-DSD) are characterized by ovarian follicles and seminiferous tubules in the same individual, with a wide range of atypical genitalia. We report on two sibs with atypical genitalia and SRY-negative 46,XX DSD, OT-DSD was confirmed only in the boy, while the girl had bilateral ovaries. Chromosome microarray analysis (CMA) showed a 737-kb duplication at Xq27.1 including the entire SOX3 gene in both sibs, which was confirmed by quantitative real time PCR. Also, X chromosome inactivation assay showed random inactivation in both sibs. Whole exome sequencing revealed no pathogenic or likely pathogenic variant. CMA of the parents showed normal results for both, suggesting that germline mosaicism could be the reason of recurrence of this duplication in the siblings. Our results support a pathogenic role of SOX3 overexpression in 46,XX subjects leading to variable DSD phenotypes.

Clinical and laboratory differences between chromosomal and undefined causes of non-obstructive azoospermia: A retrospective study

ABSTRACT BACKGROUND: Knowledge of clinical and laboratory differences between chromosomal and undefined causes aids etiological research on non-obstructive azoospermia. OBJECTIVE: Compare clinical and laboratory differences between men with non-obstructive azoospermia due to chromosomal anomalies versus undefined causes DESIGN AND SETTING: A cross-sectional retrospective study conducted at a public university hospital in Campinas (Brazil) METHODS: All men aged 20-40 years with non-obstructive azoospermia were included in the analysis. RESULTS: The 107 cases included 14 with Klinefelter syndrome (KS) (13%), 1 with mosaic KS, 4 with sex development disorders (2 testicular XX, 1 NR5A1 gene mutation, and 1 mild androgen insensitivity syndrome) (4%), 9 with other non-obstructive azoospermia etiologies (8%), and 79 with undefined causes. The 22 chromosomal anomaly cases (14 KS, 1 mosaic KS, 2 testicular XX, 4 sex chromosome anomalies, and 1 autosomal anomaly) were compared with the 79 undefined cause cases. The KS group had lower average testicular volume, shorter penile length, and lower total testosterone levels but greater height, arm span, serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels, and gynecomastia frequency (absent in the undefined group and affecting more than half of the KS group). Patients with testicular XX DSD had LH, FSH, and penile length data intermediate between the KS and undefined cause groups, testicular volume similar to the KS group, and other data similar to the undefined group. CONCLUSION: Clinical and laboratory data differentiate men with non-obstructive azoospermia and chromosomal anomalies, particularly KS and testicular XX, from those with undefined causes or other chromosomal anomalies.

Clinical and laboratory differences between chromosomal and undefined causes of non-obstructive azoospermia: A retrospective study.

BACKGROUND: Knowledge of clinical and laboratory differences between chromosomal and undefined causes aids etiological research on non-obstructive azoospermia. OBJECTIVE: Compare clinical and laboratory differences between men with non-obstructive azoospermia due to chromosomal anomalies versus undefined causes. DESIGN AND SETTING: A cross-sectional retrospective study conducted at a public university hospital in Campinas (Brazil). METHODS: All men aged 20-40 years with non-obstructive azoospermia were included in the analysis. RESULTS: The 107 cases included 14 with Klinefelter syndrome (KS) (13%), 1 with mosaic KS, 4 with sex development disorders (2 testicular XX, 1 NR5A1 gene mutation, and 1 mild androgen insensitivity syndrome) (4%), 9 with other non-obstructive azoospermia etiologies (8%), and 79 with undefined causes. The 22 chromosomal anomaly cases (14 KS, 1 mosaic KS, 2 testicular XX, 4 sex chromosome anomalies, and 1 autosomal anomaly) were compared with the 79 undefined cause cases. The KS group had lower average testicular volume, shorter penile length, and lower total testosterone levels but greater height, arm span, serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels, and gynecomastia frequency (absent in the undefined group and affecting more than half of the KS group). Patients with testicular XX DSD had LH, FSH, and penile length data intermediate between the KS and undefined cause groups, testicular volume similar to the KS group, and other data similar to the undefined group. CONCLUSION: Clinical and laboratory data differentiate men with non-obstructive azoospermia and chromosomal anomalies, particularly KS and testicular XX, from those with undefined causes or other chromosomal anomalies.

So, and if it is not congenital adrenal hyperplasia? Addressing an undiagnosed case of genital ambiguity.

BACKGROUND: The Congenital Adrenal Hyperplasia due to 21 hydroxylase deficiency is the most common cause of genital ambiguity in persons with XX sexual chromosomes. Genital ambiguity among persons with XY sexual chromosomes comprises diverse and rare etiologies. The deficiency of 17-beta-hydroxysteroid dehydrogenase type 3 enzyme (HSD17B3) is a rare autosomal recessive disorder due to functionally altered variants of the HSD17B3 gene. In this disorder/difference of sex development, the conversion of androstenedione into testosterone is impaired. The appearance of external genitalia of 46,XY individuals varies from typically male to almost female. CASE PRESENTATION: We report on a child presenting severe ambiguous genitalia. Due to access constraints, specialized care did not start until the child was 10 months old. Parents are consanguineous and were born in an area of high isonymy that is a cluster for rare recessive diseases. A new homozygous missense variant c.785G > T was found in exon 10 of the HSD17B3 gene. CONCLUSIONS: Researchers-clinicians and researchers-researchers collaborative efforts to elucidate the genetic basis of this disease were critical since this etiologic investigation is not available through the public health system. This case exemplifies the families' pilgrimage in cases of genital ambiguity due to a rare genetic condition. Recognizing the etiology was the baseline to provide information on prognosis and treatment options, and to shelter family and child doubts and hopes in order to better support their decisions.

Effective combined antiretroviral therapy provides partial immune recovery to mycobacterial antigens in vertically infected, BCG-vaccinated youth living with HIV.

BACKGROUND: We assessed the cytokine response by PBMC of youth living with HIV (YLHIV) under combined antiretroviral therapy (cART) to Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (BCG) antigens. METHODS: PBMC from 20 Brazilian YLHIV under cART with long-term (≥1 year) virological control, and 20 healthy controls were cultured for 24-96 h under stimulation with BCG, Mtb lysates, ESAT-6 and SEB. We measured TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10 and IL-17 in culture supernatants using a cytometric bead array. RESULTS: Controls had higher IFN-γ production at 24, 48, 72 and 96 h upon stimulation with BCG lysate, plateauing at 48 h (Median = 1991 vs. 733 pg/mL; p = 0.01), and after 48-72 h of stimulation with Mtb lysate, plateauing at 48 h (3838 vs. 2069 pg/mL; p = 0.049). YLHIV had higher TNF-α production at all time points upon stimulation with ESAT-6, with highest concentration at 36 h (388 vs. 145 pg/mL; p = 0.02). Within the YLHIV group, total CD4 T cell count and CD4/CD8 ratio were associated with IFN-γ response to Mtb lysate and ESAT-6, respectively. CONCLUSIONS: Even under long-term cART, YLHIV seem to have a suboptimal T-helper-1 response to mycobacterial antigens. This can be explained by early immunodeficiency in vertical infection, with lasting damage.

Differences between patients who achieved asthma control and those who remain uncontrolled after standardized severe asthma care strategy.

OBJECTIVE: To assess clinical, functional, and inflammatory patterns of children and adolescents with severe uncontrolled asthma, and investigate the differences between patients who achieved asthma control and those who remain uncontrolled after standardized asthma care strategy. METHODS: Screening all children and adolescents with asthma from the Pediatric Pulmonology Outpatient Clinic of Unicamp, Brazil, and included those with severe uncontrolled asthma according to GINA guidelines criteria. Patients were assessed at baseline and after by demographic and medication data, questionnaires (Asthma Control Test and Pediatric Asthma Quality of Life Questionnaire), Six-Minute Walk Test, skin prick test, spirometry, induced sputum, and blood collection (total immunoglobulin E and eosinophil count). Cytokine dosage was analyzed in sputum supernatant and serum by Cytometric Bead Array. RESULTS: Thirty-three patients with severe uncontrolled asthma were included (median age 10.9 [7.00-17.60] years). All patients presented satisfactory adherence to treatment and 50% of them achieved good asthma control after six-month follow-up (p < 0.001). Patients who achieved asthma control reported higher intervals since their last exacerbation episode (p = 0.008) and higher quality of life scores (p < 0.001) as compared to patients who remained uncontrolled. We found no changes in lung function markers, inflammatory biomarkers, or cytokine levels between patients with uncontrolled and controlled asthma. CONCLUSION: Participation of six months in a structured outpatient clinic for children with severe asthma had a notable improvement in control and quality of life of patients. This demonstrates the importance of a global assessment, focused on peculiarities presented by patients with severe uncontrolled asthma.

Ovarian Cancer-Associated Ascites Have High Proportions of Cytokine-Responsive CD56bright NK Cells.

Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients' blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.

Preservation of cytotoxic granule production in response to mycobacterial antigens by T-lymphocytes from vertically HIV-infected Brazilian youth on effective combined antiretroviral therapy

ABSTRACT Background: HIV infection harms adaptive cellular immunity mechanisms. Long-term virological control by combined antiretroviral therapy (cART) reduces the risk of mycobacterial infections. Thus, we aimed to study cellular responses to mycobacterial antigens in 20 HIV-infected adolescents with at least one year of virological control (HIV-RNA <40 copies/mL) and 20 healthy adolescents. Methods: We evaluated CD8 and γδ T-cell degranulation by measurement of CD107a membrane expression after stimulation with lysates from BCG (10 µg/mL) and H37RA Mycobacterium tuberculosis (Mtb, 10 µg/mL). Immune activation and antigen-presenting ability were also assessed by determination of HLA-DR, CD80, and CD86 markers. Results: TCR γδ T-cell CD107a expression was similar between groups in response to mycobacterial antigens, and lower in the HIV-infected group in response to mitogen. Higher baseline HLA-DR expression and lower mycobacterial-stimulated expression was found within the HIV-infected group. Conclusions: Similar degranulation in stimulated CD8+ and TCR γδ T-cells from HIV-infected adolescents, when compared to healthy controls suggests long-term immunological preservation with immune reconstitution under successful cART. However, differences in HLA-DR expression may represent ongoing inflammation and lower specific responses in HIV-infected youth. These features may be relevant in the context of the precocity and severity of vertically acquired HIV infection.

Three new Brazilian cases of 17α-hydroxylase deficiency: clinical, molecular, hormonal, and treatment features.

BACKGROUND: Deficiency of 17α-hydroxylase (17OHD) is a rare form of adrenal hyperplasia. Diagnosis is generally delayed, impairing appropriate treatment. CASE PRESENTATION: Here, we report the clinical, molecular, hormonal, and treatment data of three unrelated 17OHD patients, aged 14-16 years with hypergonadotrophic hypogonadism; uncontrolled hypertension; primary adrenal insufficiency; and high progesterone, low to normal potassium, and low dehydroepiandrosterone, androstenedione, and testosterone levels. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) at baseline and after an adrenocorticotropic hormone test showed low cortisol and cortisone and high deoxycorticosterone (DOC) and corticosterone levels; both DOC/21-deoxycortisol and costicosterone/cortisol ratios were very high. Patient 2 had 46,XX karyotype and patients 1 and 3, had 46,XY. A molecular analysis showed that two of the patients were homozygous for p.W406R mutation and the other patient was compound heterozygous for p.W406R and p.P428L. Hypertension was controlled only after the administration of both prednisone and mineralocorticoid antagonist. CONCLUSIONS: Hypertension in young women must lead to diagnostic suspicion, even in the pre-pubertal period. The basal level of progesterone is an indicator of 17OHD. Mineral and glucocorticoid ratios obtained from LC-MS/MS can reinforce the diagnosis. Hypertension can be controlled using glucocorticoid replacement therapy and mineralocorticoid antagonist.

408 Cases of Genital Ambiguity Followed by Single Multidisciplinary Team during 23 Years: Etiologic Diagnosis and Sex of Rearing.

Objective. To evaluate diagnosis, age of referral, karyotype, and sex of rearing of cases with disorders of sex development (DSD) with ambiguous genitalia. Methods. Retrospective study during 23 years at outpatient clinic of a referral center. Results. There were 408 cases; 250 (61.3%) were 46,XY and 124 (30.4%) 46,XX and 34 (8.3%) had sex chromosomes abnormalities. 189 (46.3%) had 46,XY testicular DSD, 105 (25.7%) 46,XX ovarian DSD, 95 (23.3%) disorders of gonadal development (DGD), and 19 (4.7%) complex malformations. The main etiology of 46,XX ovarian DSD was salt-wasting 21-hydroxylase deficiency. In 46,XX and 46,XY groups, other malformations were observed. In the DGD group, 46,XY partial gonadal dysgenesis, mixed gonadal dysgenesis, and ovotesticular DSD were more frequent. Low birth weight was observed in 42 cases of idiopathic 46,XY testicular DSD. The average age at diagnosis was 31.7 months. The final sex of rearing was male in 238 cases and female in 170. Only 6.6% (27 cases) needed sex reassignment. Conclusions. In this large DSD sample with ambiguous genitalia, the 46,XY karyotype was the most frequent; in turn, congenital adrenal hyperplasia was the most frequent etiology. Malformations associated with DSD were common in all groups and low birth weight was associated with idiopathic 46,XY testicular DSD.

Quality of sleep and quality of life in adolescents infected with human immunodeficiency virus.

OBJECTIVES: To assess sleep characteristics of adolescents infected by HIV, and to ascertain whether psychosocial aspects are associated to the quality of sleep. METHODS: A cross-sectional study assessing 102 HIV-infected adolescents of both genders, aged between 10 and 20 years-old and 120 Controls. Data collection was performed by applying the Sleep Disturbance Scale for Children, the Epworth Sleepiness Scale, and the Pediatric Quality of Life Inventory. RESULTS: A sleep disturbance prevalence of 77.4% was found in patients, and a 75% prevalence in controls, and there was correlation between quality of sleep and of life. HIV-infected adolescents scored higher for sleep breathing disorders and had higher prevalence of excessive daytime sleepiness. CONCLUSIONS: HIV-infected adolescents had similar quality of sleep compared to healthy adolescents. This may be explained by the steady improvements in daily living as a result of successful anti-retroviral therapy, and by the vulnerability that affects Brazilian adolescents living in major urban centers.

Quality of sleep and quality of life in adolescents infected with human immunodeficiency virus / Qualidade do sono e qualidade de vida em adolescentes infectados pelo vírus da imunodeficiência humana

OBJECTIVES: To assess sleep characteristics of adolescents infected by HIV, and to ascertain whether psychosocial aspects are associated to the quality of sleep. METHODS: A cross-sectional study assessing 102 HIV-infected adolescents of both genders, aged between 10 and 20 years-old and 120 Controls. Data collection was performed by applying the Sleep Disturbance Scale for Children, the Epworth Sleepiness Scale, and the Pediatric Quality of Life Inventory. RESULTS: A sleep disturbance prevalence of 77.4% was found in patients, and a 75% prevalence in controls, and there was correlation between quality of sleep and of life. HIV-infected adolescents scored higher for sleep breathing disorders and had higher prevalence of excessive daytime sleepiness. CONCLUSIONS: HIV-infected adolescents had similar quality of sleep compared to healthy adolescents. This may be explained by the steady improvements in daily living as a result of successful anti-retroviral therapy, and by the vulnerability that affects Brazilian adolescents living in major urban centers.

OBJETIVOS: Avaliar as características do sono de adolescentes infectados pelo HIV e estudar se os aspectos psicossociais estão associados à qualidade do sono. MÉTODOS: Estudo transversal, que avaliou 102 adolescentes, com idades entre 10 e 20 anos, infectados pelo HIV, e 120 controles, de ambos os gêneros. Para a coleta de dados, aplicaram-se: a Sleep Disturbance Scale for Children, a Epworth Sleepiness Scale, e o Pediatric Quality of Life Inventory. RESULTADOS: Verificou-se prevalência de distúrbios do sono em 77,4% dos pacientes e em 75% nos controles, e houve correlação entre qualidade do sono e de vida. Adolescentes HIV-positivos apresentaram maior pontuação nos distúrbios respiratórios do sono e maior prevalência de sonolência diurna excessiva. CONCLUSÕES: Adolescentes infectados pelo HIV apresentaram qualidade de sono semelhante à da população saudável. Isso provavelmente decorre pela melhora de suas condições de vida resultante do sucesso da terapia antirretroviral em pacientes HIV-positivos e pelas vulnerabilidades que afetam adolescentes brasileiros de grandes centros urbanos.

Impaired humoral response to vaccines among HIV-exposed uninfected infants.

Little is known about the vaccine protective response for infants born from HIV-infected mothers. We evaluated the antibody response to hepatitis B, tetanus, and diphtheria vaccine in vertically HIV-exposed uninfected infants and compared them to those of control infants not exposed to the virus. The quantitative determination of specific neutralizing antibodies against hepatitis B, diphtheria, and tetanus were performed blindly on serum samples. The results showed that 6.7% of the HIV-exposed uninfected individuals were nonresponders to hepatitis B vaccine (anti-HBs titer, <10 mIU/ml), and 64.4% were very good responders (anti-HBs titer, ≥1,000 mIU/ml), whereas only 3.6% of the nonexposed infants were nonresponders (χ(2)=10.93; 1 df). The HIV-exposed uninfected infants showed protective titers for diphtheria and tetanus but lower geometric mean anti-tetanus titers compared to those of the HIV-unexposed infants. Our data point to the necessity of evaluating vaccine immune responses in these children and reinforced that alterations in lymphocyte numbers and functions reported for newborns from HIV-infected mothers interfere with the vaccine response.

Immunogenicity of a whole-cell pertussis vaccine with low lipopolysaccharide content in infants.

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gammadelta-positive (gammadelta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gammadelta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gammadelta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gammadelta(+) cell expansions were similar with the two vaccines.