Non-Conjugated Chitosan-Based Nanoparticles to Proteic Antigens Elicit Similar Humoral Immune Responses to Those Obtained with Alum.

Biodegradables Chitosan-based Nanoparticles (CS NPs) have been extensively studied as delivery system for therapeutic molecules and as efficient carriers or adjuvants in experimental vaccination. Physicochemical association between CS NPs and antigens is a key step for the biological function as carrier devices. However, for the adjuvant CS NPs property, it is not well known if coupling with vaccine antigens is required or not to potentiate the immune response. To address this issue, in this work, we evaluated the potential adjuvant effect of CS NPs by simply mixing with two different antigens such as Bovine Serum Albumin (BSA) or E protein from Dengue Virus serotype 2 (E protein DENV2). Thus the CS NPs were prepared by ionic gelation with sodium tripolyphosphate, resulting particles among 68 and 188 nm of size. Immunization of 6­8 week old female BALB/c mice, were carried out by intraperitoneal route with a simple combination of CS NPs either with BSA (CS NPs-BSA) at 10 µg or with E protein DENV2 (CS NPs-Protein E) at 5 µg. Combinations with the above antigens with CS NPs elicited robust specific primary and secondary humoral responses comparable to alum, a well-known adjuvant. BSA-specific IgG titers were detectable by day 14 after priming with the CS NPs-BSA formulation, with titers that ranged from 102 to 103 EU ml-. After a second immunization, the anti-BSA titers ranged around 104 EU ml-. In contrast, in the group of mice immunized with the protein alone, BSA-specific serum IgG titers were undetectable at day 14 and 28. For the immunizations with the CS NPs-E protein formulation, we observed also a remarkable specific-antibody production in the primary response, with titers reaching 103 EU ml-. After the booster immunization the anti-E protein DENV2 antibodies titers reached peak values around 104 EU ml-. Interestingly, for both antigens, the combination with CS NPs polarized the immune response to a Th2-like profile, which is characterized mainly by the production of the IgG1 Isotype, confirming that CS NPs can enhance and modulate the humoral immune responses against different antigens independently of physicochemical conjugation. This could represent a simplification in the use of CS NPs as adjuvants in vaccination.

Plasma protein and supplemental isoleucine in milk replacers for dairy calves.

We measured the effects of milk replacers containing 0, 33, 66, or 100% of the total replaceable whey protein as bovine plasma protein (PP), without or with Ile supplementation, on the intake, growth, and health of 124 male Holstein calves for 35d. Milk replacers were formulated to contain 18% crude protein and 20% fat, with contents of Lys and Met equalized. When fed to calves at 1.5% of body weight (dry matter basis) under thermoneutral conditions, diets were predicted to allow average daily gains of 0.55kg/d based on metabolizable energy or 0.40kg/d based on apparent digestible protein. Protein supply was more limiting than energy so that differences in protein use could be detected. Dry matter intakes decreased with increased PP, irrespective of Ile supplementation. Final body weights decreased linearly with increasing PP, regardless of Ile supplementation. Average daily gain tended to be affected in a quadratic manner as PP increased, either with or without Ile supplementation; average daily gain and gain-feed ratio were greatest for calves fed diets containing 33% PP and lowest for calves fed 100% PP. The analyzed Lys content in the milk replacers was variable compared with formulated values, and this may have affected growth results. However, the gain-Lys ratio was affected by an interaction of the linear effect of increasing PP with Ile supplementation: it decreased with increasing PP but was improved by supplementation with Ile for calves fed 100% PP. Body measurements decreased with increasing PP inclusion; only decreased heart girth was reversed with Ile supplementation. The lowest and highest inclusion of PP, regardless of Ile supplementation, decreased the occurrence of scours compared with the control diet (all whey protein). Calves fed the lowest and highest PP without Ile supplementation also had fewer total days of scours in the first 21d. In addition, calves fed 100% PP without supplementation of Ile had fewer days of medication compared with the control diet. Even at the highest PP inclusion, average daily gain was minimally affected if Ile was supplemented. Growth rates, gain-feed ratio, and gain-Lys ratio were decreased at higher PP inclusion, but Ile overcame part of the reduction in gain-Lys ratio for 100% PP. Additional titration studies will have to be conducted to determine optimal PP inclusion rates, with a focus on supplementation of potentially limiting essential AA, as well as effects at higher growth rates.

Differences in prevalence of community-associated MRSA and MSSA among U.S. and non-U.S. born populations in six New York Community Health Centers.

BACKGROUND: Staphylococcus aureus is the most common cause of Skin and Soft Tissue Infections (SSTIs) in the community in the United States of America. Community Health Centers (CHC) serve as primary care providers for thousands of immigrants in New York. METHODS: As part of a research collaborative, 6 New York City-area CHCs recruited patients with SSTIs. Characterization was performed in all S. aureus isolates from wounds and nasal swabs collected from patients. Statistical analysis examined the differences in wound and nasal cultures among immigrant compared to native-born patients. RESULTS: Wound and nasal specimens were recovered from 129 patients and tested for antibiotic susceptibility. 40 patients were immigrants from 15 different countries. Although not statistically significant, immigrants had lower rates of MRSA infections (n = 15) than did native-born participants, and immigrants showed significantly higher rates of MSSA wound cultures (n = 11) (OR = 3.5, 95% CI: 1.3, 9.7). CONCLUSIONS: In our study, immigrants were more likely to present with SSTIs caused by MSSA than US-born patients. Immigants also reported lower frequencies of antibiotic prescription or consumption in the months prior to SSTI infection. This suggests that antibiotic resistance may vary regionally and that immigrants presenting with SSTIs may benefit from a broader range of antibiotics.

Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog.

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.

Digestive enzymes of eight Amazonian teleosts with different feeding habits.

The levels of amylase, maltase, lipase and alkaline protease from eight fish species of the Amazon were analysed. The enzyme levels are not related to fish feeding habits, reflecting their ever-changing habitats and their opportunistic feeding behaviour.

Prolonged Hya-disparate skin graft survival in ethanol-consuming mice: correlation with impaired delayed hypersensitivity.

BACKGROUND: Ethanol consumption impairs cell-mediated immunity and enhances humoral immunity. Among cell-mediated immune reactions, little is known of the effect of ethanol on chronic graft rejection. Allograft responses against the male-specific minor histocompatibility antigen, Hya, are widely used to study chronic graft rejection. METHODS: Female C57BL/6 (B6) mice were fed ethanol-containing liquid diets, were pair-fed an isocaloric liquid control diet, or were fed solid diet and water ad libitum. One week after diet initiation, the mice were grafted with split thickness, orthotopic male tail skin grafts, and the integrity of the grafts was monitored as the diet continued. Delayed hypersensitivity (DTH) was also determined in these same mice. In addition, Hya-cytolytic T-cell-deficient syngeneic major histocompatibility complex mutant B6.C-H2bm13 (bm13) and B6.C-H2bm14 (bm14) mice were assessed for skin graft rejection, DTH, and cytotoxic T-lymphocyte (CTL) activity. RESULTS: Ethanol-consuming female B6 mice are impaired in their ability to reject syngeneic male skin grafts and to develop Hya-specific DTH responses. To address the underlying mechanism, we show that Hya graft rejection correlates with DTH and not with CTL activity. Female B6 mice clearly differ from female bm13 and bm14 mice in their ability to generate CTLs against Hya antigen. Despite their inability to make Hya-specific CTL responses, bm13 and bm14 female mice, nevertheless, make Hya-specific DTH responses and ultimately reject Hya-disparate skin grafts, indicating that Hya-specific graft rejection results from DTH. Ethanol, by impairing Hya-specific DTH, inhibits Hya-specific skin graft rejection. CONCLUSIONS: We demonstrate that ethanol consumption impairs Hya-specific graft rejection. In addition, experiments with mice unable to generate anti-Hya CTLs support previous observations suggesting that DTH responses are sufficient to cause rejection of Hya-incompatible grafts.

Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) bind specifically to duplex DNA and provide a strategy for site-directed modification of genomic DNA. Recently we demonstrated TFO-mediated targeted gene knockout following systemic administration in animals. However, a limitation to this approach is the requirement for a polypurine tract (typically 15-30 base pairs (bp)) in the target DNA to afford high affinity third strand binding, thus restricting the number of sites available for effective targeting. To overcome this limitation, we have investigated the ability of chemically modified TFOs to target a short (10 bp) site in a chromosomal locus in mouse cells and induce site-specific mutations. We report that replacement of the phosphodiester backbone with cationic phosphoramidate linkages, either N,N-diethylethylenediamine or N,N-dimethylaminopropylamine, in a 10-nucleotide, psoralen-conjugated TFO confers substantial increases in binding affinity in vitro and is required to achieve targeted modification of a chromosomal reporter gene in mammalian cells. The triplex-directed, site-specific induction of mutagenesis in the chromosomal target was charge- and modification-dependent, with the activity of N,N-diethylethylenediamine > N,N-dimethylaminopropylamine phosphodiester, resulting in 10-, 6-, and <2-fold induction of target gene mutagenesis, respectively. Similarly, N,N-diethylethylenediamine and N,N-dimethylaminopropylamine TFOs were found to enhance targeting at a 16-bp G:C bp-rich target site in a chromatinized episomal target in monkey COS cells, although this longer site was also targetable by a phosphodiester TFO. These results indicate that replacement of phosphodiester bonds with positively charged N,N-diethylethylenediamine linkages enhances intracellular activity and allows targeting of relatively short polypurine sites, thereby substantially expanding the number of potential triplex target sites in the genome.

Manipulating the mammalian genome by homologous recombination.

Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies--chimeric nucleases and triplex-forming oligonucleotides--for stimulating recombination in cells.

Triplex-induced recombination in human cell-free extracts. Dependence on XPA and HsRad51.

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 x 10(-)5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51.

Specific mutations induced by triplex-forming oligonucleotides in mice.

Triplex-forming oligonucleotides (TFOs) recognize and bind to specific duplex DNA sequences and have been used extensively to modify gene function in cells. Although germ line mutations can be incorporated by means of embryonic stem cell technology, little progress has been made toward introducing mutations in somatic cells of living organisms. Here we demonstrate that TFOs can induce mutations at specific genomic sites in somatic cells of adult mice. Mutation detection was facilitated by the use of transgenic mice bearing chromosomal copies of the supF and cII reporter genes. Mice treated with a supF-targeted TFO displayed about fivefold greater mutation frequencies in the supF gene compared with mice treated with a scrambled sequence control oligomer. No mutagenesis was detected in the control gene (cII) with either oligonucleotide. These results demonstrate that site-specific, TFO-directed genome modification can be accomplished in intact animals.

Triapine (3-aminopyridine-2-carboxaldehyde- thiosemicarbazone): A potent inhibitor of ribonucleotide reductase activity with broad spectrum antitumor activity.

Previous studies from our laboratories have shown that (a) Triapine() is a potent inhibitor of ribonucleotide reductase activity and (b) hydroxyurea-resistant L1210 leukemia cells are fully sensitive to Triapine. In an analogous manner, Triapine was similarly active against the wild-type and a hydroxyurea-resistant subline of the human KB nasopharyngeal carcinoma. Triapine was active in vivo against the L1210 leukemia over a broad range of dosages and was curative for some mice. This agent also caused pronounced inhibition of the growth of the murine M109 lung carcinoma and human A2780 ovarian carcinoma xenografts in mice. Optimum anticancer activity required twice daily dosing due to the duration of inhibition of DNA synthesis which lasted about 10 hr in L1210 cells treated with Triapine in vivo. DNA synthesis in normal mouse tissues (i.e. duodenum and bone marrow) uniformly recovered faster than that in L1210 leukemia cells, demonstrating a pharmacological basis for the therapeutic index of this agent. Triapine was more potent than hydroxyurea in inhibiting DNA synthesis in L1210 cells in vivo, and the effects of Triapine were more pronounced. In addition, the duration of the inhibition of DNA synthesis in leukemia cells from mice treated with Triapine was considerably longer than in those from animals treated with hydroxyurea. Combination of Triapine with various classes of agents that damage DNA (e.g. etoposide, cisplatin, doxorubicin, and 1-acetyl-1,2-bis(methylsulfonyl)-2-(2-chloroethyl)hydrazine) resulted in synergistic inhibition of the L1210 leukemia, producing long-term survivors of tumor-bearing mice treated with several dosage levels of the combinations, whereas no enhancement of survival was found when Triapine was combined with gemcitabine or cytosine arabinoside. The findings demonstrate the superiority of Triapine over hydroxyurea as an anticancer agent and further suggest that prevention by Triapine of repair of DNA lesions created by agents that damage DNA may result in efficacious drug combinations for the treatment of cancer.

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells.

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.

Alcohol consumption alters antigen-specific Th1 responses: mechanisms of deficit and repair.

Among the physiological effects associated with excessive alcohol consumption are alterations in immune function. Alcohol impairs T-helper 1 lymphocyte (Th1) regulated, cell-mediated immune responses. Antibody responses, regulated by T-helper 2 lymphocyte (Th2), are either unimpaired or enhanced. Antigen presenting cells are central to the development of both Th1 and Th2 regulated immune responses. We used both T-cell receptor transgenic and conventionally immunized mice to demonstrate that ethanol consumption directly affects antigen presenting cells that, in turn, determines whether Th1 or Th2 response patterns predominate. Ethanol consumption inhibits Th1-associated interleukin-12 and interferon-gamma cytokine production and delayed-type hypersensitivity. Administration of exogenous recombinant interleukin-12 both restores interferon-gamma levels and delayed-type hypersensitivity responses in ethanol-consuming mice.

Triplex targets in the human rhodopsin gene.

We have explored the application of triplex technology to the human rhodopsin gene, which encodes a G-protein-linked receptor involved in the genetic disorder autosomal dominant retinitis pigmentosa (ADRP). Our results support the hypothesis that most human genes contain high-affinity triplex sites and further refine the rules governing identification and successful targeting of triplex-forming oligonucleotides (TFOs) to these sites. Using a computer search for sites 15 nucleotides in length and greater than 80% purine, we found 143 distinct sites in the rhodopsin gene and comparable numbers of sites in several other human genes. By applying more stringent criteria, we selected 17 potential target sites in the rhodopsin gene, screened them with a plasmid binding assay, and found 8 that bound TFOs with submicromolar affinity (Kd = 10(-)9-10(-)7 M). We compared purine (GA) and mixed (GT) TFOs at each site, and found that GA-TFOs consistently bound with higher affinity, and were less sensitive to pyrimidine interruptions in the target strand. High G-content favored high-affinity binding; only sites with >54% G-content bound TFOs with Kd

Glutathione levels in antigen-presenting cells modulate Th1 versus Th2 response patterns.

Current thinking attributes the balance between T helper 1 (Th1) and Th2 cytokine response patterns in immune responses to the nature of the antigen, the genetic composition of the host, and the cytokines involved in the early interaction between T cells and antigen-presenting cells. Here we introduce glutathione, a tripeptide that regulates intracellular redox and other aspects of cell physiology, as a key regulatory element in this process. By using three different methods to deplete glutathione from T cell receptor transgenic and conventional mice and studying in vivo and/or in vitro responses to three distinct antigens, we show that glutathione levels in antigen-presenting cells determine whether Th1 or Th2 response patterns predominate. These findings present new insights into immune response alterations in HIV and other diseases. Further, they potentially offer an explanation for the well known differences in immune responses in "Th1" and "Th2" mouse strains.

Triplex-directed modification of genes and gene activity.

Oligonucleotides offer enormous potential for manipulating gene function in cells and, as such, constitute a promising new class of pharmaceutical agents. Oligonucleotides that form triple helices (triplexes) at specific DNA sequences in defined genes can be used to reduce transcription selectively, to introduce site-specific mutations or to stimulate gene-specific targeted recombination.

Interleukin-12 therapy restores cell-mediated immunity in ethanol-consuming mice.

Previous studies from our laboratory show that ethanol consumption impairs antigen-specific, cell-mediated, but not, humoral immune responses of C57BL/6, BALB/c, and DO11.10 T-cell receptor transgenic mice. This ethanol-associated deficit is associated with decreased interleukin (IL)-12 and interferon-gamma (IFN-gamma) production, but not IL-2 or antigen-specific T-cell proliferation by explanted leukocytes from ethanol-consuming mice. IL-12 expression by macrophage/monocytes is viewed as a requirement for the production of IFN-gamma by Th1 lymphocytes that mediate cellular immunity. In this study, we restored antigen-specific, cell-mediated immunity, delayed hypersensitivity, to ethanol-consuming C57BL/6 or BALB/c mice with a single 100 ng of intravenous injection of recombinant IL-12 at the time of immunization. The addition of exogenous recombinant IL-12 to co-cultures of antigen-presenting cells derived from ethanol-consuming mice and purified T cells derived from ethanol-nonconsuming DO11.10 repairs the ability of Th1 cells to make IFN-gamma in response to antigen. Administration of recombinant IL-12 opens a potential for restoring cell-mediated immune function to ethanol-consuming individuals.

Development of a radioimmunoassay for Cebus apella prolactin.

Prolactin (PRL) is a pituitary hormone that plays important roles in mammalian reproductive physiology, specially lactation. The regulation of PRL secretion shows important species differences. To study PRL regulation in a subhuman primate, the Cebus apella, we developed an heterologous radioimmunoassay using an antibody against rhesus PRL (anti-m5PRL) and a Cebus apella pituitary extract as PRL standard. The assay has a sensitivity that allows measurements of cebus PRL in small amounts of Cebus apella plasma obtained from animals in different physiological conditions. Plasma cebus PRL concentrations (+/-SEM) varied in different reproductive stages. PRL concentration in adult Cebus apella females that have regular menstrual cycles (161.6 +/- 15.0 mIU/ml) was similar to that found in adult (100.3 +/- 7.6 mIU/ml) and prepuberal males (101.2 +/- 3.9 mIU/ml). PRL concentration was higher in pregnant (677.8 +/- 11.8 mIU/ml) and in nursing (625.0 +/- 47.0 mIU/ml) Cebus apella females than in 15-d post-partum non-nursing (369.0 +/- 19.0 mIU/ml) and cycling females. PRL concentration in Cebus apella newborns (719.0 +/- 49.2 mIU/ ml) was similar to that found in pregnant and nursing females, and higher than in the other females as well as adult and prepuberal males. These differences in PRL concentration in different physiological conditions are similar to that observed in humans and other primates. A PRL response to thyrotropin releasing hormone (TRH) was demonstrated in 2 nursing Cebus apella females, similar to the response found in nursing woman and rhesus. Altogether, the data presented support the proposal that the assay developed to measure PRL in Cebus apella is an adequate tool to study the physiology of PRL in this species.