Introduction

The synthesis of prolactin (PRL) is not limited to the pituitary as many other extra-pituitary tissues, including T lymphocytes can produce PRL.^{1, 2, 3} This hormone has a prominent immunomodulatory role both in humoral and cell-mediated immune responses by acting at the endocrine, paracrine and autocrine levels.^{4, 5, 6, 7} Under physiological conditions the production of PRL by the pituitary is mainly under the control of a tonic inhibitory mechanism mediated by dopamine (DA) acting through dopamine D₂ receptors in the lactotrophs;⁸ however, the mechanisms controlling the production of PRL in extra-pituitary sites remains largely unknown. This seems to be a relevant issue, as hypophysectomized rats show a postoperative rise in serum PRL concentrations and depend on this residual PRL for survival.⁹ In other words, it seems that when the main source of PRL production is abolished, extra-pituitary PRL could be exported into the general circulation and accumulated through endocytosis by other cells^{10, 11} to fulfil important biological actions.

Animal and human studies have demonstrated that PRL functions as a necessary comitogen for different cell types, including human T and B cells,^{12, 13} and by acting through the PRL receptors present in these cells^{14, 15} PRL stimulates both cell proliferation and survival. Furthermore, PRL facilitates the progression of the cell cycle, both in PRL-dependent and PRL-independent cell lines^{13, 16} and is considered to be an anti-apoptotic hormone as opposed to glucocorticoids.^{16, 17, 18}

Critically ill patients receiving a DA infusion had a marked reduction in serum PRL levels simultaneously with an immediate, although transitory, decrease in the in vitro T-cell response to Concanavalin-A.¹⁹ This, together with the recent demonstration that the DA concentration within human lymphocytes (which depends both on lymphocyte synthesis and uptake of extracellular DA) functions as an autocrine loop whereby lymphocytes downregulate their own proliferative activity,²⁰ highlights the importance of exploring, directly or indirectly, the possible interplay within human lymphocytes of PRL and DA, both in healthy and ill humans.

Despite the overwhelming information regarding the possible role of PRL, especially biologically active PRL, in HIV infection, this area has remained largely unexplored. Nevertheless, some studies have suggested indirectly that the existence of a low DA tone in men with HIV-infection^{21, 22, 23, 24} could represent an adaptive mechanism attempting to stimulate human lymphocyte proliferation at a maximum possible rate in an attempt to survive.²⁵ The aim of the present study was to explore the in vivo and in vitro production of bioactive PRL in a group of untreated HIV-infected men and its association with the cell cycle and apoptosis of PBMC in these patients.

Materials and Methods

Subjects

Twenty adult (26.9 6.3 years) euthyroid volunteer men with HIV infection (group 1) classified as category C2 (n = 3) and C3 (n = 17)²⁶ recently diagnosed and naive to any type of specific anti-retroviral treatment were studied. Their CD4⁺ T-lymphocyte count was 104 112 10⁶/L. The viral load determined in 14 patients using the Nuclisens HIV-1 QT kit (Organon Teknika, Boxtel, The Netherlands), a nucleic acid sequence based amplification (NASBA) assay,²⁷ was 36 700 14 125 RNA copies/mL of plasma (range 1800–160 000 RNA copies/mL of plasma, except for one patient with 1.1 10⁶ RNA copies/mL of plasma). These patients were compared to 14 adult (24.0 1.8 years) euthyroid HIV-negative volunteer men (group 2) with a CD4⁺ T-lymphocyte count of 689 208 10⁶/L. All men in group 2 and 17 men from group 1 had a normal body weight for height according to Mexican standards;²⁸ the remaining three patients were considered to have AIDS wasting syndrome. None of the 34 volunteers had been regularly ingesting any medications known to influence serum PRL concentrations. The diagnosis of HIV infection was confirmed by immunoblot analysis for HIV antibodies, and T cells expressing the CD4 and CD8 molecules were determined by three-colour flow cytometry using fluorescent specific monoclonal antibodies.²⁹ Detailed clinical characteristics of the volunteers have been previously published.²⁴

The protocol was approved by the Review Board and the Human Ethical Committee of the Hospital General de Puebla, Coordinación de los Servicios Médicos de Salud of the Health Department in Puebla, México and a written informed consent was obtained from all subjects who volunteered to participate in the study. The study was carried out according to the principles of the Declaration of Helsinki.

Experimental protocol

All volunteers were identically studied after a 10–12 h overnight fasting. Three morning basal blood samples were obtained at 15 min intervals and thereafter every 30 min for 2 h, following a 10 mg i.v. bolus of metoclopramide (Carnotprim, Laboratorios Carnot, Mexico City).²⁴ No physical activity, smoking or sleeping was allowed during the test. None of the volunteers, specifically those of group 1, had any side-effects, particularly extra-pyramidal symptoms, following metoclopramide administration.

Cell preparation and cultures

Human PBMC were obtained at sampling time –30 min by Ficoll-Hypaque density gradient³⁰ of heparinized venous blood obtained from uninfected and HIV-infected men. Peripheral blood mononuclear cells were recovered from the interfase and cultured at a density of 2 10⁵ cells/0.15 mL in serum-free culture medium (AIM-V, Life Technologies, Grand Island, NY, USA) in the absence and in the presence of a previously determined mitogenic concentration of Concanavalin-A (Con-A), at a final concentration of 1.5 g/mL, which was previously found to be optimal to activate cell cycling. The viability of cells, as determined by propidium iodide exclusion, was always above 95%. Cells were incubated for 72 h at 37°C in a humidified 95% air/5% CO₂ atmosphere. In a small sample of patients (n = 6) and controls (n = 7) an aliquot of the culture medium was kept at –70°C until analysed for PRL bioactivity.

Cell cycle and apoptosis

After completion of the culture period, cells were harvested, transferred to 12 75 polypropylene tubes and washed with PBS. Cells were then permeabilized and stained for total nuclear DNA with a mixture of ribonuclease and propidium iodide (PI), with the aid of commercial reagents (DNA-Prep, Beckman Coulter, Miami, FL, USA). Flow cytometry was performed in an EPICS Elite ESP instrument (Coulter, Miami, FL, USA), equipped with an air-cooled argon laser to excite fluorescence at 480 nm, while the emission of PI was collected at 675 nm. Cell doublets were discriminated through gating events whose integral and peak fluorescence signals kept a linear proportion, and a minimum of 5000 gated events were collected from each aliquot. Raw cytometric data were analysed by the Multicycle® software (Phoenix Flow Systems, San Diego, CA, USA), which enables estimation of proportions of cells in each phase of the cell cycle. Due to the loss of low molecular weight DNA, cells undergoing apoptosis appear as a sub-G₀/G₁ Gaussian peak and may be quantified simultaneously.³¹ Apoptosis was determined in 19 HIV-infected men and in 13 uninfected control men.

Immunoreactive prolactin (RIA-PRL)

Serum RIA-PRL was measured in duplicate in each sample throughout the study using commercially available kits (Ortho Clinical Diagnostics Ltd, Amersham, UK). The characteristics of the assay have been previously published.²⁴

Bioactive prolactin (BIO-PRL)

The Nb2 cell line was originally obtained from Dr PW Gout (Department of Cancer Endocrinology, British Columbia Cancer Agency, Vancouver, Canada). Prolactin bioactivity (BIO-PRL) was measured using the Nb2 lymphoma cell assay as described by Tanaka et al.³² with minor modifications. Briefly, cells were kept at 37°C in Fischer's medium containing 10% fetal bovine serum (FBS) as a source of lactogen, 10% horse serum (HS), 10^–4 mol/L 2-mercaptoethanol, 50 IU/mL penicillin and 50 g/mL streptomycin. The cells were arrested in the early G₁ phase of the cell cycle by pre-incubation (18–24 h) in lactogen-free medium. At this time, resumption of the cell cycle was stimulated by the addition of increasing concentrations of recombinant human PRL (r-hPRL). Cultures per duplicate were further maintained in an atmosphere of 95% air/5% CO₂ at 37°C for 72 h.

Prolactin-like bioactivity was assayed in aliquots of serum samples (obtained throughout the metoclopramide test) and of culture medium from PBMC at different dilutions to ascertain parallelism with the standard curve. The effect of hPRL and PRL-like substance from serum or PBMC medium on cell proliferation was analysed by the cell proliferation kit II (XTT, Boerhinger Mannheim, Mannheim, Germany). The intra- and interassay coefficients of variation for the Nb2 bioassay were 3.4% and 6.2%, respectively. Serum samples for normal subjects and patients were assayed at dilution 1:50. The dynamic range of the bioassay was from 3.9 to 500 pg/mL and the sensitivity was 1.9 pg/mL.

Statistical analysis

The RIA and BIO-PRL concentrations of the three basal serum samples were pooled and expressed as mean basal serum concentration. The areas under the curve (AUC) were calculated for RIA-PRL and BIO-PRL.³³ Values in the text and figures represent mean ( SD). The two-tailed Student's t-test for unpaired samples was used for between-group comparisons. A P 0.05 was considered statistically significant. For the analysis of the bioactive PRL-like production by cultured PBMC, the Wilcoxon matched-pairs signed-rank test was used for within group comparisons and the Mann–Whitney U-test for between-group comparisons. Correlations between variables were analysed using the Pearson's correlation coefficient. The approach to the correlation analysis between the viral load and serum PRL was to fit first and second degree polynomial equations to the independent variables (SPSS version 6.01, SPSS Inc, Chicago, IL, USA). The regression equation with the highest correlation coefficient was selected, as long as it fulfilled the statistical criterium of significance at a level of P 0.01.

Results

Serum immunoreactive prolactin (RIA-PRL)

Mean basal RIA-PRL concentrations were similar in HIV infected (group 1) and uninfected (group 2) men (7.3 4.4 cf. 7.8 3.3 ng/mL, respectively). The AUC RIA-PRL in group 1 was 5690 1387 ng/mL per 120 min and 9168 1321 ng/mL per 120 min in group 2 (P < 0.0001).

Serum bioactive prolactin (BIO-PRL)

Mean basal BIO-PRL concentration in group 1 was 6.1 2.6 ng/mL and in group 2 was 4.4 1.9 ng/mL (P = 0.03), and the AUC BIO-PRL were 2057 280 ng/mL per 120 min for group 1 compared to 2526 298 ng/mL per 120 min for group 2 (P = 0.001).

Bioactive/immunoreactive prolactin ratio

Mean basal BIO/RIA PRL ratio was greater in group 1 than in group 2 (0.96 0.39 cf. 0.59 0.22, respectively; P = 0.001). Equally, the AUC BIO/RIA ratio during 120 min was greater in group 1 (0.40 0.15) than in group 2 (0.28 0.04) (P = 0.001).

Cell cycle

Under non-stimulated conditions group 1 had a lower percentage of PBMC in G₀/G₁ phases than group 2 (97.0 2.1 cf. 98.4 1.0, respectively; P = 0.01) and a greater percentage of cells in S phase (2.6 1.9 cf. 0.78 0.8, respectively; P < 0.001) without significant differences in the percentage of cells in G₂/M phases (0.60 0.6 cf. 0.92 0.6, respectively). After stimulation with Con-A in group 1 and group 2 there was a quantitatively similar and significant (P < 0.001) increase in the percentage of PBMC in S phase (12.2 6.6 cf. 12.7 6.9, respectively) and in G₂/M phases (3.7 2.6 cf. 3.5 3.0, respectively), without significant differences between groups.

Apoptosis

The percentage of PBMC undergoing apoptosis was 9.97 7.07 in group 1 as compared to 3.93 1.75 in group 2 (P =0.002). In group 1, 17 patients had <200 CD4⁺ T lymphocytes and in 14 of these patients the apoptotic cells were 7.0%; in the remaining three patients together with the three patients with 200 CD4⁺ T lymphocytes, the percentage of apoptotic cells was 6.9%. All individuals in group 2 had a percentage of apoptotic cells 6.9%.

Production of bioactive prolactin by cultured PBMC

In the group 1 sample the basal or non-stimulated in vitro production of bioactive PRL by PBMC (34.0 9.1 pg/mL) decreased after Con-A stimulation (7.0 3.5 pg/mL) (P = 0.01; Figure 1)

Figure 1.

In vitro production of bioactive prolactin (PRL) by cultured PBMC in HIV-infected men (group 1) and in uninfected control men (group 2). Non-stimulated condition (A); with Concanavalin-A (Con-A) stimulation (B). The results are expressed as pg/mL of recombinant human prolactin (r-hPRL). Values represent mean SD.

Full figure and legend (14K)

In contrast, in the group 2 sample the basal in vitro production of bioactive PRL (20.6 11.4 pg/mL) increased after Con-A stimulation (58.4 37.7 pg/mL) (P = 0.01). Basal production of bioactive PRL was greater in group 1 than in group 2 (P = 0.04), while the opposite occurred after Con-A stimulation (P = 0.004).

Correlations

Mean basal BIO-PRL concentrations had a positive linear correlation with the percentage of non-stimulated PBMC in S + G₂/M phases (r = 0.366, P = 0.03; Figure 2a), while the latter had a negative linear correlation with the absolute number of CD4⁺ T lymphocytes in uninfected, but not in HIV infected men (r = –0.534, P = 0.04; Figure 2b). The percentage of apoptotic cells in HIV-infected men was most closely correlated with the absolute number of CD4⁺ T lymphocytes using an inverse regression model (Figure 3). Considering both groups, the percentage of apoptotic cells had a negative linear correlation with the AUC RIA-PRL 120 min (Figure 4) and a positive linear correlation with the AUC BIO/RIA PRL ratio 120 min (Figure 5). Considering only HIV-infected men (group 1), the negative linear correlation between the percentage of apoptotic cells and the AUC RIA-PRL 120 min remained significant (r = – 0.3572, P < 0.0001), as did the positive linear correlation with the AUC BIO/RIA–PRL ratio 120 min (r = 0.4580, P = 0.04; data not shown).

Figure 2.

(a) Correlation between mean basal bioactive prolactin (BIO-PRL) and the percentage of non-stimulated PBMC in S + G₂/M phases of the cell cycle in HIV-infected men () and in uninfected control men (). (b) Correlation between the percentage of non-stimulated PBMC in S + G₂/M phases and the CD4⁺ T-lymphocyte count in uninfected control men (), but not in HIV-infected men ().

Full figure and legend (32K)

Figure 3.

Correlation between the percentage of apoptotic PBMC and the CD4⁺ T-lymphocyte count in HIV-infected men.

Full figure and legend (12K)

Figure 4.

Correlation between the percentage of apoptotic PBMC and the area under the serum immunoreactive prolactin curve (AUC RIA-PRL) 120 min in response to i.v. metoclopramide in HIV-infected men () and in uninfected control men ().

Full figure and legend (18K)

Figure 5.

Correlation between the percentage of apoptotic PBMC and the area under the curve for the bioactive- immunoreactive prolactin ratio (AUC BIO/RIA ratio 120 min) following i.v. metoclopramide in HIV-infected men () and in uninfected control men ().

Full figure and legend (15K)

Discussion

The present findings in serum RIA-PRL and especially in BIO-PRL concentrations suggest that these HIV-infected men had a diminished hypothalamic DA tone, as previously suggested,^{23, 24} because their mean basal BIO-PRL concentration was greater and the RIA-PRL and BIO-PRL responses to intravenous metoclopramide were lower than in uninfected men.^{34, 35}

The mechanisms controlling the extra-pituitary production of PRL (specifically, in human lymphocytes) are unclear; however, this hormone acts as a comitogen for human T and B cells^{12, 13} and the intralymphocyte DA concentration is known to downregulate their own proliferation in humans.²⁰ Furthermore, a temporal decrease in Con-A stimulated lymphocyte proliferation has been described in critically ill patients with low serum PRL concentrations induced by DA infusions.¹⁹ Thus, the observed greater in vitro production of bioactive PRL by non-stimulated PBMC of HIV infected men, could be, at least partially, taken as indirect evidence of the existence of a low intralymphocyte DA concentration (tone?), which would favour lymphocyte proliferation via autocrine, paracrine and/or endocrine mechanisms.^{5, 20} This point of view is supported by the finding in HIV-infected men of a smaller percentage of non-stimulated PBMC in resting (G₀/G₁) phases and a higher percentage in S phase, along with the significant correlation between the latter and the serum basal BIO-PRL (Figure 2a). Whether or not within human lymphocytes DA and PRL could be interacting in a similar fashion, as in the hypothalamic-pituitary region, can not be demonstrated from the present study.

Interestingly, the negative correlation between the percentage of non-stimulated PBMC in S + G₂/M phases and CD4⁺ T lymphocytes only observed in uninfected men raises the possibility that a decrease in the CD4⁺ T-lymphocyte count is associated with progression of the cell cycle toward the facilitation of lymphocyte proliferation. This mechanism appears to be disrupted or non-operative in HIV-infected men (Figure 2b).

The paradoxical effect of Con-A upon the in vitro production of a bioactive PRL-like substance in HIV-infected men suggests that their non-stimulated PBMC were already at a maximal PRL-producing capacity and a further stimulation may downregulate the specific receptor and/or interrupt the PRL–PRL receptor signal transduction.⁷ Consequently, the lack of significant differences between groups 1 and 2 after Con-A stimulation in the percentage of PBMC at each phase of the cell cycle, indicates that the non-stimulated PBMC of these HIV-infected men may already be under maximal PRL stimulation, and could not respond to further mitogenic stimulation.

The findings on apoptosis and serum PRL deserve particular consideration. The percentage of apoptotic cells in HIV-infected men was strongly associated in a negative fashion with the CD4⁺ T-lymphocyte count and with the AUC RIA-PRL 120 min. One possible explanation is that as apoptosis increased in the HIV-infected men, the decrease in CD4⁺ T lymphocytes paralleled a diminution in the DA tone of the body (as shown by the lower serum RIA-PRL response to the i.v. metoclopramide)^{23, 24, 35} attempting to maintain PRL synthesis and release as high as possible.²⁵ At the same time, increasing apoptosis was associated in a positive fashion with the AUC BIO/RIA PRL 120 min, which suggests that augmented apoptosis was paralleled by an increased availability of BIO- over RIA-PRL, which is consistent with the anti-apoptotic properties of this hormone.^{16, 17, 18} Undoubtedly there are many other factors influencing apoptosis that were not considered here, but PRL does appear to be important.

Overall, these observations are in agreement with the proposal that PRL may regulate T-cell proliferation, serving as a third growth factor.¹³ Recently, the metoclopramide-induced rise in serum PRL has been shown to stimulate splenocyte proliferation and splenocyte IL-2 and IL-3 release in rats after trauma haemorrhage.³⁶ Furthermore, extracellular PRL seems to be a requisite for the IL-2-driven T-lymphocyte proliferation, by its action at the nucleus level.¹¹

In summary, it is suggested that the existence of a diminished DA tone (both hypothalamic, and perhaps intralymphocytic)^{23, 24} in these patients may be serving as an adaptive mechanism that is attempting to maintain the highest possible BIO-PRL availability (whether of pituitary or lymphocytic origin) to promote the proliferation of CD4⁺ T lymphocytes,^{13, 25} in an attempt to survive. In this context, and based on recent publications, the possibility of using recombinant human PRL as an adjunct therapeutic approach in HIV patients does not seem inappropriate,^{25, 37} especially if one considers its pleiotropic effects and limited toxicity after systemic administration in animals.³⁸

References

1. Sinha YN. Structural variants of prolactin: Occurrence and physiological significance. Endocr. Rev. 1995; 16: 354–69. | Article | PubMed | ISI | ChemPort |
2. Ben-Jonathan N, Mershon JL, Allen DL, Steinmetz RW. Extrapituitary prolactin: Distribution, regulation, functions, and clinical aspects. Endocr. Rev. 1996; 17: 639–69. | Article | PubMed | ChemPort |
3. Matera L. Action of pituitary and lymphocyte prolactin. Neuroimmunomodulation 1997; 4: 171–80. | PubMed | ChemPort |
4. Reber M. Prolactin and immunomodulation. Am. J. Med. 1993; 95: 637–44. | Article | PubMed |
5. Matera L. Endocrine, paracrine and autocrine actions of prolactin on immune cells. Life Sci. 1996; 59: 599–614. | Article | PubMed | ISI | ChemPort |
6. Yu-Lee LY. Molecular actions of prolactin in the immune system. Proc. Soc. Exp. Biol. Med. 1997; 215: 35–52. | PubMed | ChemPort |
7. Clevenger CV, Freier DO, Kline JB. Prolactin receptor signal transduction in cells of the immune system. J. Endocrinol. 1998; 157: 187–97. | Article | PubMed | ISI | ChemPort |
8. Ben-Jonathan N. Dopamine: a prolactin-inhibiting hormone. Endocr. Rev. 1985; 6: 564–89. | PubMed | ChemPort |
9. Nagy E, Berczi I. Hypophysectomized rats depend on residual prolactin for survival. Endocrinology 1991; 128: 2776–84. | PubMed | ChemPort |
10. Kooijman R, Hooghe-Peters EL, Hooghe R. Prolactin, growth hormone and insulin-like growth factor-1 in the immune system. Adv. Immunol. 1996; 63: 377–454. | PubMed | ISI | ChemPort |
11. Clevenger CV, Rusell DA, Appasamy PM, Prystowsky MB. Regulation of interleukin 2-driven T-lymphocyte proliferation by prolactin. Proc. Natl Acad. Sci. USA 1990; 87: 6460–4. | Article | PubMed | ChemPort |
12. Rusell DH, Matrisian L, Kibler R, Larson DF, Poulus B, Magun BE. Prolactin receptors in human lymphocytes and their modulation by cyclosporine. Biochem. Biophys. Res. Commun. 1984; 21: 899–906.
13. Clevenger CV, Sillman AL, Hanley-Hyde J, Pristowsky MB. Requirement for prolactin during cell cycle regulated gene expression in cloned T-lymphocytes. Endocrinology 1992; 130: 3216–22. | Article | PubMed | ChemPort |
14. Pellegrini I, Lebrun JJ, Ali S, Kelly PA. Expression of prolactin and its receptor in human lymphoyd cells. Mol. Endocrinol. 1992; 6: 1023–31. | Article | PubMed | ISI | ChemPort |
15. Dardenne M, de Morales MCL, Kelly PA, Gagnerault MC. Prolactin receptor expression in human hematopoietic tissues analyzed by flow cytofluorometry. Endocrinology 1994; 134: 2108–14. | Article | PubMed | ISI | ChemPort |
16. Leff MA, Buckley DJ, Krumenacker JS, Reed JC, Miyashita T, Buckely AR. Rapid modulation of the apoptosis regulatory genes, bcl-2 and bax by prolactin in rat Nb2 lymphoma cells. Endocrinology 1996; 137: 5456–62. | Article | PubMed | ChemPort |
17. Krumenacker JS, Buckley DJ, Leff MA et al. Prolactin- regulated apoptosis of Nb2 lymphoma cells: pim-1, bcl-2, and bax expression. Endocrine 1998; 9: 163–70. | Article | PubMed | ChemPort |
18. Weimann E, Baixeras E, Zamzani N, Kelly P. Prolactin blocks glucocorticoid induced cell death by inhibiting the disruption of the mitochondrial membrane. Leuk. Res. 1999; 23: 751–62. | Article | PubMed | ISI | ChemPort |
19. Devins SS, Miller A, Herndon BL, O'Toole L, Reisz G. Effects of dopamine on T-lymphocyte proliferative responses and serum prolactin concentrations in critically ill patients. Crit. Care Med. 1992; 20: 1644–9. | PubMed | ChemPort |
20. Bergquist J, Tarkowski A, Ekman R, Ewing A. Discovery of endogenous catecholamines in lymphocytes and evidence for catecholamine regulation of lymphocyte function via an autocrine loop. Proc. Natl Acad. Sci. USA 1994; 91: 12 912–6.
21. Berger JR, Kumar M, Kumar A, Fernández JB, Levin B. Cerebrospinal fluid dopamine in HIV-1 infection. AIDS 1994; 8: 67–71. | PubMed | ChemPort |
22. Montero A, Fernández MA, Cohen JE, Luraghi MR, Sen L. Prolactin levels in the cerebrospinal fluid of patients with HIV infection and AIDS. Neurol. Res. 1998; 20: 2–4. | PubMed | ChemPort |
23. Parra A, Ramirez-Peredo J, Coutiño B et al. Impaired metoclopramide-induced pituitary prolactin release in men with human immunodeficiency virus infection. J. Lab. Clin. Med. 1999; 133: 70–4. | Article | PubMed | ChemPort |
24. Parra A, Ramírez-Peredo J, Larrea F et al. Decreased endogenous dopaminergic tone and increased basal bioactive prolactin in men with human immunodeficieny virus infection. Clin. Endocrinol. (in press).
25. Parra A, Ramírez-Peredo J. The uncoupled couple? Prolactin and CD4 lymphocytes in HIV infection. Med. Hypotheses 1999; 53: 425–8. | Article | PubMed | ChemPort |
26. Centers for disease control and prevention. 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Morbid Mortal Week Rep 1999; 41: 1–19.
27. Segondy M, Ly TD, Lapeyre M, Montes B. Evaluation of the Nuclisens HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 RNA levels in plasma. J. Clin. Microbiol. 1998; 36: 3372–4. | PubMed | ChemPort |
28. Casillas LE, Vargas LA. Height and weight charts for Mexican adults. Arch. Invest. Méd. (Méx.) 1980; 11: 157–74. | ChemPort |
29. Ruiz-Argüelles A. Flow cytometry in the clinical laboratory. Principles, applications and problems. Clin. Chim. Acta 1992; 11: 513–27.
30. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest 1968; 21: 77–98. | PubMed | ChemPort |
31. Darzynkiewicz Z, Li Gong J. Assays in cell viability. Discrimination of cells dying by apoptosis. In: Darzynkiewicz Z, Robins JP, Crissman HA (eds). Methods in Cell Biology: Flow Cytometry. San Diego: Academic Press, 1994: 15–38.
32. Tanaka T, Shiu RPC, Gout PW, Beer CT, Noble RL, Friesen HG. A new sensitive and specific bioassay for lactogenic hormone measurement of prolactin and growth hormone in human serum. J. Clin. Endocrinol. Metab. 1980; 51: 1058–63. | PubMed | ChemPort |
33. Zenobi PA, Jaeggi-Groisman SE, Riesen WE, Roder ME, Froesch ER. Insulin-like growth factor-1 improves glucose and lipid metabolism in type 2 diabetes mellitus. J. Clin. Invest. 1992; 90: 2234–41. | PubMed | ChemPort |
34. Quigley ME, Judd SJ, Gilliland GB, Yen SSC. Effects of a dopamine antagonist on the release of gonadotropin and prolactin in normal women and in women with hyperprolactinemic anovulation. J. Clin. Endocrinol. Metab. 1979; 48: 718–20. | PubMed | ChemPort |
35. Parra A, Barrón J, Marín VA, Coutiño B, Belmont J, Coria I. Acute dopaminergic blockade augments the naloxone-induced LH rise in estrogen-treated postmenopausal women. Maturitas 1997; 27: 91–9. | Article | PubMed | ChemPort |
36. Knoferl MW, Angell MK, Ayala A, Cioffi WG, Bland KI, Chandry I. Insight into the mechanism by which metoclopramide improve immune functions after trauma-hemorrhage. Am. J. Physiol. Cell Physiol. 2000; 79: C72–80.
37. Richards SM, Murphy WJ. Use of human prolactin as a therapeutic protein to potentiate immunohematopoietic function. J. Neuroimmunol. 2000; 109: 56–62. | Article | PubMed | ChemPort |
38. Woody MA, Welniak LA, Richards S et al. Use of neuroendocrine hormones to promote reconstitution after bone marrow transplantation. Neuroimmunomodulation 1999; 6: 69–80. | Article | PubMed | ISI | ChemPort |

Acknowledgements

This work was partially supported by grants from the Special Programme of Research, Development and Research Training in Human Reproduction of the World Health Organization (Geneva, Switzerland) and the Consejo Nacional de Ciencia y Tecnología (Conacyt, México). Dr Víctor Cabrera was the recipient of a Research Training Grant from the Latin American Program of Research and Training in Human Reproduction (PLACIRH, México). We are indebted to the patients and the uninfected control individuals for their participation in the study. We thank Dr Irma Coria and Miss Marcela Zambrano for their valuable assistance in the statistical analysis.