Pregnant women
The epidemiological features of the mothers entering the study are presented in Table
1. In the control group, only one mother was a smoker. Yet, smoking and drug abuse (marihuana, cocaine or crack) was common among the HIV+ ones. Both maternal groups had a similar age and a similar time of gestation. HIV+ mothers had a cesarean section in all but one case. Among HIV-negative mothers 11 had a vaginal and 4 a cesarean delivery. Antiretroviral therapy included protease inhibitors in all but 3 cases. Mean time of maternal HAART treatment was 5 months. Eight mothers got pregnant while already using HAART. HIV viral load was undetectable in 23 HIV+ mothers and low in the remaining thirteen (Table
1).
Table 1
Epidemiologic characteristics of the mothers (median and range)
Age at delivery (years) | 29 (18 - 41) | 32 (20 - 39) |
Weeks of gestation | 39.7 (36 - 42) | 38.2 (36 - 42) |
Smoker only | 1 | 5 |
Drug user only | 0 | 3 |
Smoker and drug user | 0 | 6 |
Non-smoker non-drug user | 14 | 22 |
Days of HAART use | - | 154 (49 - 273) |
detectable viral load copies/mL* | - | 496 (54 - 36586) |
Mothers' PB counts are shown on Table
2. HIV+ mothers had hemoglobin values similar to that of HIV-uninfected ones, but showed a significantly higher mean corpuscular volume (MCV), lower total leukocyte and neutrophil counts. The proportion of T CD4+ and T CD8+ lymphocytes of the HIV+ mothers presented similar values in smokers, non-smokers and drug users.
Table 2
Peripheral blood features of the mothers at 35 weeks of pregnancy (median and range)
RBC* (M/μL) | 3.9 | 3.3 | <0.005 |
| (3.7 - 4.7) | (2.5 - 4.7) | |
HB* (g/dL) | 11.7 | 10.7 | 0.09 |
| (9.6 - 13.9) | (9.0 - 13.7) | |
MCV* (fL) | 88 | 104 | <0.005 |
| (72 - 94) | (75 - 118) | |
WBC* (K/μL) | 8.2 | 7.2 | 0.04 |
| (3.2 - 12.2) | (3.0 - 15.0) | |
Total neutrophils (K/μL) | 5.7 | 4.86 | 0.07 |
| (2.3 - 8.8) | (2.2 - 10.9) | |
Total lymphocytes (K/μL) | 1.8 | 1.6 | 0.94 |
| (0.6 - 2.7) | (0.5 - 4.8) | |
CD4+ count | n.d.* | 511 | |
| | (57 - 1189) | |
CD8+ count | n.d.* | 676 | |
| | (321 - 1979) | |
CD4/CD8 ratio | n.d.* | 0.73 | |
| | (0.06 - 1.97) | |
Platelets (K/μL) | 200 | 217 | 0.96 |
| (102 - 392) | (99 - 360) | |
The cytokine concentrations in the supernatant of PBMC cultures not stimulated and after stimulation with BCG and PHA in mothers at 35 weeks of gestation are presented on Table
3. HIV+ mothers showed lower values of IL-4 and IL-7, but higher values of TNFα in non-stimulated cultures. Production of IL-2 and IL-12 by PBMCs in non-stimulated as well as stimulated cultures were similar both in normal and HIV-infected mothers.
Table 3
Cytokine levels (in pg/ml, median and range) without antigenic stimulus and after stimulation with BCG and PHA in the supernatant from mothers' peripheral mononuclear cell cultures collected at 35 weeks of gestation
IL-2 non-stimulated | 0.0 (0 -76) | 0.0 (0 - 767) | 0.47 |
BCG | 0.0 (0 - 78) | 2.14 (0 - 232) | 0.44 |
PHA | 89.8 (0 -968) | 24.5 (0 - 1421) | 0.34 |
IL-4 non-stimulated | 41.0 (38 - 278) | 22.2 (9.2 - 76) | <0.005 |
BCG | 39.1 (37 - 216) | 22.8 (9.5 - 76) | <0.005 |
PHA | 52.0 (39 - 146) | 30.14 (14 - 419) | <0.005 |
IL-7 non-stimulated | 67.0 (0 -88) | 0.0 (0 - 90) | 0.05 |
BCG | 66.0 (0 - 89) | 0.0 (0 - 86) | 0.04 |
PHA | 66.0 (0 - 85) | 0.0 (0 - 87) | 0.03 |
IL-10 non-stimulated | 0.0 (0 - 490) | 17 (0 - 18015) | 0.14 |
BCG | 736 (0 - 4910) | 764 (0 - 43384) | 0.49 |
PHA | 498 (0 - 2925) | 405 (0 - 58344) | 0.56 |
IL-12 non-stimulated | 0.51 (0 - 2.83) | 0.51 (0 -2.4) | 0.62 |
BCG | 1.18 (1 - 31) | 1.0 (0 - 11) | 0.27 |
PHA | 1.34 (0 - 51) | 1.53 (0 - 33) | 0.68 |
IFNγ non-stimulated | 0.0 (0 - 94) | 13.2 (0 - 131) | 0.73 |
BCG | 2.73 (0 - 1343) | 29.2 (0 - 999) | 0.42 |
PHA | 110.2 (0 - 2528) | 308.9 (0 - 5143) | 0.16 |
TNFα non-stimulated | 245.4 (0 - 4826) | 526.8 (0 - 7928) | <0.0005 |
BCG | 5374.0 (0 - 13852) | 2072.7 (86 - 34130) | 0.83 |
PHA | 827.6 (261 - 20889) | 1513.3 (244 - 14448) | 0.22 |
The CD4/CD8 ratio in PB of HIV+ mothers showed an inverse relation with viral load (r = -0.30; p = 0.02) and production of TNFα by PBMCs (non-stimulated) (r = -0.27; p = 0.04) and a direct correlation with IL-10 (r = 0.35; p = 0.01). There was no correlation with IL-7.
In a multiple regression, we obtained the formula:
Maternal CD4/CD8 ratio = 0.946 + 5.6 × 10-5 × IL-10 - 9.8 × 10-5 × TNFα - 0.0567 × log viral load + 0.9468 R2 = 0.335 ; p = 0.004
The stability of the model was confirmed in a bootstrap resampling procedure. Among 100 new models, "log viral load" entered in 97%, "IL-10" level in 89%, "TNFα" in 60% and "IL-7" in 28% of them.
Newborns
The birth data of the newborns are described in Table
4. Children of HIV+ mothers had a similar height but a lower weight than those born to uninfected mothers. Furthermore, they had lower hemoglobin levels, a higher MCV and lower leukocyte and neutrophil counts than non-exposed newborns. The number of days of maternal ARV treatment during pregnancy did not influence these values, but there was a good correlation between maternal and cord blood red blood cells (RBCs) (r = 0.56; p < 0.0005) and MCV (r = 0.54; p < 0.0005). This was less evident concerning hemoglobin values (r = 0.21; p = 0.06).
Table 4
Birth data, cord blood counts and lymphocyte subsets of the newborns (median and range)
Weight (grams) | 3100 | 2962 | <0.005 |
| (2988 - 3915) | (1885 - 3885) | |
Height (cm) | 48 | 48 | 0.87 |
| (40 - 53) | (33 - 53) | |
Capurro score | 278 | 267 | <0.005 |
| (256 - 294) | (255 - 293) | |
RBC (M/μL) | 4.6 | 3.5 | <0.005 |
| (4.1 - 5.0) | (2.7 - 4.7) | |
HB (g/dL) | 15.7 | 13.7 | <0.005 |
| (13.2 - 18.0) | (9.1 - 16.4) | |
MCV (fL) | 104 | 117 | <0.005 |
| (99 - 124) | (88 - 150) | |
Platelets (K/μL) | 202 | 242 | 0.12 |
| (119 - 328) | (103 - 455) | |
WBC (K/μL) | 11.1 | 9.1 | 0.08 |
| (4.4 - 19.5) | (4.0 - 22.0) | |
Total neutrophils (K/μL) | 4.2 | 3.5 | 0.32 |
| (0.8 - 9.4) | (1.0 - 8.8) | |
Total lymphocytes (K/μL) | 5.7 | 5.1 | 0.16 |
| (3.0-9.1) | (1.5 - 10.5) | |
% CD3+ | 18.3 | 24.8 | 0.03 |
| (13.8 - 32.3) | (12.0 - 41.5) | |
% CD3/CD4+ | 12.6 | 17.2 | 0.32 |
| (8.9 - 23.3) | (3.0 - 28.2) | |
% CD3+/CD8+ | 4.6 | 6.4 | 0.002 |
| (1.8 - 10.7) | (2.3 - 15.5) | |
CD4/CD8 ratio | 3.5 | 2.3 | 0.04 |
| (1.6 - 8.3) | (0.2 - 6.5) | |
% CD19+ | 3.6 | 5.7 | 0.01 |
| (2.0 - 5.9) | (1.7 - 16.2) | |
% CD19+CD5+ ** | 59.5 | 75.8 | 0.006 |
| (42.8 - 91.1) | (47.7 - 97.0) | |
The birth weight of HIV-exposed infants showed a relation with the duration of maternal HAART treatment during pregnancy and the TNFα production (non-stimulated cultures) in supernatants of cultures of PB mononuclear cells collected at the end of gestation. Maternal age, smoking, weeks of gestation and child's gender had no relation to birth weight.
In a multiple regression we obtained the formula:
Birth weight = 3327 - 1.48 × days of HAART - 5 × unstimulated maternal TNFα p = 0.006, R2 = 0.216 ( CI 95% : 0.215 - 0.383 )
The bootstrap resampling study confirmed this result: "days of HAART treatment" appeared in 77% and "non-stimulated maternal TNFα at 35 weeks of gestation" was present in 70% of the new data sets. But, "weeks of gestation" appeared in 40%, "maternal smoking" in 38%, "mother's age" in 12% and "child's gender" in 10% of the new data sets.
The distribution of the several lymphocyte subsets in cord blood is shown on Table
4. The percentage of CD3+ lymphocytes in HIV-exposed newborns was increased when compared with the non-exposed ones. The CD4/CD8 ratio was significantly lower.
In HIV-exposed neonates the relation of the number of cord T lymphocytes with maternal age, days of HAART use during pregnancy, smoking and drug abuse was examined in a multiple regression. For T CD8+ cell no model could be obtained.
The percentage of T CD4+ cells was lower in children from older and smoking mothers.
This percentage could be calculated by the formula:
Cord % T CD4+ cells = -0.428 × mother's age (years) - 4.91 (S) + 30.52;
S = 1 for smoking and 0 for non-smoking mother.
R2 = 0.242 (CI 95%: 16.6 - 51.1)
Drug abuse and days of maternal HAART treatment during pregnancy did not enter the model. The stability of the model for cord blood T CD4+ cells was confirmed in the bootstrap resampling. Among 100 new models, "maternal age" entered in 87% and "maternal smoking" in 72% of them. "Duration of maternal HAART treatment" entered in 42%, and "drug abuse" in 11% of the models.
CD3, CD4 and CD8 immunophenotyping was carried out in cell cultures of cord blood from 22 newborns from HIV + mothers and 10 non-exposed ones. Median background CD3+ proliferation was 9.6% (0.5-25.9) in the HIV+ group and 10.4% (6.2-91.7) in the controls (p = 0.44). The blast percentage in PHA-stimulated cultures showed a median of 40.3% (1.5-71.6) for the HIV+ group and 54.2 (11.7-74.8) for the non-exposed controls. The proportion of PHA-proliferating CD4+ and CD8+ cells were not different between the groups (Table
5).
Table 5
Percentages of CD4+ and CD8+ blastic cells determined by flow cytometry in CBMCa cultures incubated with PHAb from 22 newborns from HIV+ mothers and 10 HIV negative controls (median and range)
CD4+ | 88.6 | 86.3 | 0.807 |
| (3.4-97.2) | (1.7-98.3) | |
CD8+ | 6.2 | 7.6 | 1.00 |
| (1.4-79.5) | (0.3-92.7) | |
Cord blood CD19+ lymphocytes (Table
6) were increased in newborns of HIV+ mothers due to an increase of CD19+/CD5+ cells. Children of drug users had the highest numbers of total B lymphocytes. The duration of HAART during pregnancy showed no correlation with the total number of cord blood B-cells or with that of CD19+/CD5+ ones.
Table 6
B lymphocyte subsets and B-cell maturation in cord blood studied in the CD45/CD34/CD19/CD22 and sIgM/CD34/CD19/CD10 combinations (median and range)
% CD19+
| 3.6 | 5.2 | 7.0 | 0.01 |
% CD19+CD5+ * | 59.5 | 77.8 | 67.6 | 0.007 |
% CD45lowCD34+CD19+CD22-/+ ** | 0.73 | 0.42 | 1.1 | |
| (0.2 - 3.13) | (0.07 - 1.22) | (0.78 - 1.4) | 0.01 |
% CD45-/+CD34lowCD19+CD22+ *** | 2.62 | 2.94 | 13.0 | |
| (0.24 - 6.8) | (1.5 - 8.2) | (8.8-19.4) | 0.01 |
% CD45+CD34-CD19+CD22+ **** | 96.7 | 96.7 | 86.3 | |
| (91 - 99) | (91.6 - 98.4) | (79 - 90) | 0.02 |
%sIgM-CD34+CD19+CD10-/+ ** | 0.4 | 0.2 | 1.5 | |
| (0.10-2.2) | (0.05-0.95) | (0.4-2.7) | 0.05 |
%sIgM+CD34-/+CD19+CD10-/+ *** | 2.4 | 2.2 | 6.5 | |
| (0.87-7.1) | (0.6-4.5) | (2.1-71) | 0.13 |
%sIgM+CD34-CD19+CD10-/+ **** | 97.1 | 97.3 | 92.5 | |
| (91.3-98.9) | (94.6-99.3) | (92.0-95.0) | 0.05 |
B-cell maturation was analyzed in two four-color combinations (Table
6). The CD45/CD34/CD19/CD22 combination was the most useful to reveal an increase in immature and intermediary B-precursors in infants of HIV+ mothers. This feature was more pronounced in children of drug users.
In HIV-exposed neonates, there was no significant difference in the proportion of immature cells with the newborn's gender (p = 0.33) or children of smokers and non-smokers (p = 0.21), but those of drug users showed a higher percentage (p = 0.007) (Table
6). There was no significant correlation between this variable and gestation duration or maternal age but with the number of days of maternal HAART use (r = 0.53; p = 0.01).
In a multiple regression, examining the relation between the percentage of cord blood immature cells with maternal drug abuse and the number of days of HAART treatment during pregnancy, the proportion of immature/intermediate (IMAT) B cells could be calculated by the formula:
% Cord IMAT B cells = 0.02 × days of HAART + B + 0.72; R2 = 0.859 (CI - 0.653 - 0.943)
With B = 12.0 for drug abusers and B = 0 for all other mothers.
The stability of this regression model was tested by the bootstrap resampling study, where both variables entered in 98% of the new data sets.
The cytokine concentrations in the supernatant of CBMC cultures (Table
7) showed a high correlation with the values found for their mothers (Table
3), excluding those of IFNγ and TNFα. IL-4 and IL-7 production was lower in HIV+ mothers and cord blood mononuclear cells. Concerning IL-10, there was no significant difference between HIV+ and HIV negative mothers. However, HIV-exposed newborns produced higher amounts than non-exposed, although these differences had a low statistical significance.
Table 7
Cytokine levels (in pg/ml, median and range) without antigenic stimulus and after stimulation with BCG and PHA in the supernatant of cord blood cell cultures
IL-2 non-stimulated | 0.0 (0 -76) | 0.72 (0 - 81) | 0.13 |
BCG | 0.0 (0 -78) | 0.36 (0 - 79) | 0.54 |
PHA | 43.7 (0 - 1278) | 18.9 (0 - 5936) | 0.46 |
IL-4 non-stimulated | 39.2 (39 - 76) | 25.5 (10 - 76) | <0.005 |
BCG | 39.4 (40 - 76) | 25.6 (9 - 76) | <0.005 |
PHA | 39.2 (39 - 78) | 26.7 (9.5 - 76) | <0.005 |
IL-7 non-stimulated | 66.0 (0 - 87) | 0.0 (0 - 87) | 0.13 |
BCG | 66.4 (0 - 87) | 1.5 (0 - 93) | 0.24 |
PHA | 66.5 (0 - 87) | 0.0 (0.0 - 88) | 0.11 |
IL-10 non-stimulated | 0 (0 - 88) | 3.5 (0 - 1028) | 0.13 |
BCG | 199 (0 - 1522) | 582 (0 - 7049) | 0.06 |
PHA | 224 (0 - 1934) | 410 (0 - 36093) | 0.19 |
IL-12 non-stimulated | 0.66 (0 - 13.3) | 0.24 (0 - 5.7) | 0.41 |
BCG | 0.83 (0 - 26) | 0.74 (0 - 3.64) | 0.10 |
PHA | 0.62 (0 - 3) | 1.1 (0 - 8) | 0.58 |
IFNγ non-stimulated | 0.0 (0 - 66) | 17.5 (0 - 194) | 0.01 |
BCG | 0.0 (0 - 1188) | 19.4 (0 - 550) | 0.02 |
PHA | 24.3 (0 - 2445) | 73.2 (0 - 2928) | 0.28 |
TNFα non-stimulated | 136.4 (0 - 500) | 181.6 (0 - 3381) | 0.66 |
BCG | 1458.1 (138 - 7026) | 1069.0 (283 -14724) | 0.96 |
PHA | 535.0 (298 - 5672) | 658.4 (0 - 6313) | 0.63 |
Concerning TNFα, HIV+ mothers had a higher production, especially drug users (p = 0.01). CBMCs of HIV-exposed infants produced similar amounts of TNFα but more IFNγ than the control group. No correlation was found between the number of days of maternal HAART treatment during pregnancy and CBMC cytokine production.
In non-exposed newborns, no correlation was found between the percentage of CD19+ cells as well as CD19+/CD5+ ones and any cytokine production in CBMCs. However, in HIV-exposed newborns, an inverse correlation was found between total cord blood CD19+ cells and cord IL-4 and IL-7 production in non-stimulated cultures (Table
8).
Table 8
Correlations found in HIV-exposed neonates between cord blood B-cell subsets and cytokine production by cord mononuclear cells in culture
IL-4 non-stimulated | r = - 0.49; p = 0.001 | r = 0.25; p = 0.07 |
BCG | r = - 0.46; p = 0.002 | n.s.* |
PHA | r = - 0.40; p = 0.008 | n.s.* |
IL-7 non-stimulated | r = - 0.46; p = 0.002 | r = 0.32; p = 0.02 |
BCG | r = - 0.39; p = 0.01 | r = 0.41; p = 0.006 |
PHA | r = - 0.48; p = 0.001 | r = 0.45; p = 0.003 |
IL-10 non-stimulated | r = - 0.31; p = 0.05** | r = 0.33; p = 0.02 |
BCG | r = - 0.48; p = 0.02** | r = 0.51; p < 0.0005 |
PHA | r = - 0.39; p = 0.04** | r = 0.41; p = 0.03 |
A positive correlation was seen between CD19+/CD5+ cells and cord IL-7 and IL-10, but no correlation was found between these cells and production of IL-4, IFNγ and TNFα by CBMCs.