The single-cell opioid responses in the context of HIV (SCORCH) consortium.

Substance use disorders (SUD) and drug addiction are major threats to public health, impacting not only the millions of individuals struggling with SUD, but also surrounding families and communities. One of the seminal challenges in treating and studying addiction in human populations is the high prevalence of co-morbid conditions, including an increased risk of contracting a human immunodeficiency virus (HIV) infection. Of the ~15 million people who inject drugs globally, 17% are persons with HIV. Conversely, HIV is a risk factor for SUD because chronic pain syndromes, often encountered in persons with HIV, can lead to an increased use of opioid pain medications that in turn can increase the risk for opioid addiction. We hypothesize that SUD and HIV exert shared effects on brain cell types, including adaptations related to neuroplasticity, neurodegeneration, and neuroinflammation. Basic research is needed to refine our understanding of these affected cell types and adaptations. Studying the effects of SUD in the context of HIV at the single-cell level represents a compelling strategy to understand the reciprocal interactions among both conditions, made feasible by the availability of large, extensively-phenotyped human brain tissue collections that have been amassed by the Neuro-HIV research community. In addition, sophisticated animal models that have been developed for both conditions provide a means to precisely evaluate specific exposures and stages of disease. We propose that single-cell genomics is a uniquely powerful technology to characterize the effects of SUD and HIV in the brain, integrating data from human cohorts and animal models. We have formed the Single-Cell Opioid Responses in the Context of HIV (SCORCH) consortium to carry out this strategy.

EcoHIV Infection of Primary Murine Brain Cell Cultures to Model HIV Replication and Neuropathogenesis.

BACKGROUND: EcoHIV is a chimeric HIV that replicates in mice in CD4+ T cells, macrophages, and microglia (but not in neurons), causing lasting neurocognitive impairment resembling neurocognitive disease in people living with HIV. The present study was designed to develop EcoHIV-susceptible primary mouse brain cultures to investigate the indirect effects of HIV infection on neuronal integrity. RESULTS: We used two EcoHIV clones encoding EGFP and mouse bone marrow-derived macrophages (BMM), mixed mouse brain cells, or enriched mouse glial cells from two wild-type mouse strains to test EcoHIV replication efficiency, the identity of productively infected cells, and neuronal apoptosis and integrity. EcoHIV replicated efficiently in BMM. In mixed brain cell cultures, EcoHIV targeted microglia but did not cause neuronal apoptosis. Instead, the productive infection of the microglia activated them and impaired synaptophysin expression, dendritic density, and axonal structure in the neurons. EcoHIV replication in the microglia and neuronal structural changes during infection were prevented by culture with an antiretroviral. CONCLUSIONS: In murine brain cell cultures, EcoHIV replication in the microglia is largely responsible for the aspects of neuronal dysfunction relevant to cognitive disease in infected mice and people living with HIV. These cultures provide a tool for further study of HIV neuropathogenesis and its control.

CCL2 is required for initiation but not persistence of HIV infection mediated neurocognitive disease in mice.

HIV enters the brain within days of infection causing neurocognitive impairment (NCI) in up to half of infected people despite suppressive antiretroviral therapy. The virus is believed to enter the brain in infected monocytes through chemotaxis to the major monocyte chemokine, CCL2, but the roles of CCL2 in established NCI are not fully defined. We addressed this question during infection of conventional and CCL2 knockout mice with EcoHIV in which NCI can be verified in behavioral tests. EcoHIV enters mouse brain within 5 days of infection, but NCI develops gradually with established cognitive disease starting 25 days after infection. CCL2 knockout mice infected by intraperitoneal injection of virus failed to develop brain infection and NCI. However, when EcoHIV was directly injected into the brain, CCL2 knockout mice developed NCI. Knockout of CCL2 or its principal receptor, CCR2, slightly reduced macrophage infection in culture. Treatment of mice prior to and during EcoHIV infection with the CCL2 transcriptional inhibitor, bindarit, prevented brain infection and NCI and reduced macrophage infection. In contrast, bindarit treatment of mice 4 weeks after infection affected neither brain virus burden nor NCI. Based on these findings we propose that HIV enters the brain mainly through infected monocytes but that resident brain cells are sufficient to maintain NCI. These findings suggest that NCI therapy must act within the brain.

Treatment with buprenorphine prior to EcoHIV infection of mice prevents the development of neurocognitive impairment.

Approximately 15-40% of people living with HIV develop HIV-associated neurocognitive disorders, HAND, despite successful antiretroviral therapy. There are no therapies to treat these disorders. HIV enters the CNS early after infection, in part by transmigration of infected monocytes. Currently, there is a major opioid epidemic in the United States. Opioid use disorder in the context of HIV infection is important because studies show that opioids exacerbate HIV-mediated neuroinflammation that may contribute to more severe cognitive deficits. Buprenorphine is an opioid derivate commonly prescribed for opiate agonist treatment. We used the EcoHIV mouse model to study the effects of buprenorphine on cognitive impairment and to correlate these with monocyte migration into the CNS. We show that buprenorphine treatment prior to mouse EcoHIV infection prevents the development of cognitive impairment, in part, by decreased accumulation of monocytes in the brain. We propose that buprenorphine has a novel therapeutic benefit of limiting the development of neurocognitive impairment in HIV-infected opioid abusers as well as in nonabusers, in addition to decreasing the use of harmful opioids. Buprenorphine may also be used in combination with HIV prevention strategies such as pre-exposure prophylaxis because of its safety profile.

Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders.

HIV-associated neurocognitive disorders (HAND) have been linked to dysregulation of glutamate metabolism in the central nervous system (CNS) culminating in elevated extracellular glutamate and disrupted glutamatergic neurotransmission. Increased glutamate synthesis via upregulation of glutaminase (GLS) activity in brain immune cells has been identified as one potential source of excess glutamate in HAND. However, direct evidence for this hypothesis in an animal model is lacking, and the viability of GLS as a drug target has not been explored. In this brief report, we demonstrate that GLS inhibition with the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) can reverse cognitive impairment in the EcoHIV-infected mouse model of HAND. However, due to peripheral toxicity DON is not amenable to clinical use in a chronic disease such as HAND. We thus tested JHU083, a novel, brain penetrant DON prodrug predicted to exhibit improved tolerability. Systemic administration of JHU083 reversed cognitive impairment in EcoHIV-infected mice similarly to DON, and simultaneously normalized EcoHIV-induced increases in cerebrospinal fluid (CSF) glutamate and GLS activity in microglia-enriched brain CD11b + cells without observed toxicity. These studies support the mechanistic involvement of elevated microglial GLS activity in HAND pathogenesis, and identify JHU083 as a potential treatment option. Graphical Abstract Please provide Graphical Abstract caption.Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders .

Intranasal insulin therapy reverses hippocampal dendritic injury and cognitive impairment in a model of HIV-associated neurocognitive disorders in EcoHIV-infected mice.

OBJECTIVE: Almost half of HIV-positive people on antiretroviral therapy have demonstrable mild neurocognitive impairment (HIV-NCI), even when virologically suppressed. Intranasal insulin therapy improves cognition in Alzheimer's disease and diabetes. Here we tested intranasal insulin therapy in a model of HIV-NCI in EcoHIV-infected conventional mice. DESIGN AND METHODS: Insulin pharmacokinetics following intranasal administration to mice was determined by ELISA. Mice were inoculated with EcoHIV to cause NCI; 23 days or 3 months after infection they were treated daily for 9 days with intranasal insulin (2.4 IU/mouse) and examined for NCI in behavioral tests and HIV burdens by quantitative PCR. Some animals were tested for hippocampal neuronal integrity by immunostaining and expression of neuronal function-related genes by real time-quantitative PCR. The effect of insulin treatment discontinuation on cognition and neuropathology was also examined. RESULTS: Intranasal insulin administration to mice resulted in µIU/ml levels of insulin in cerebrospinal fluid with a half-life of about 2 h, resembling pharmacokinetic parameters of patients receiving 40 IU. Intranasal insulin treatment starting 23 days or 3 months after infection completely reversed NCI in mice. Murine NCI correlated with reductions in hippocampal dendritic arbors and downregulation of neuronal function genes; intranasal insulin reversed these changes coincident with restoration of cognitive acuity, but they returned within 24 h of treatment cessation. Intranasal insulin treatment reduced brain HIV DNA when started 23 but not 90 days after infection. CONCLUSION: Our preclinical studies support the use of intranasal insulin administration for treatment of HIV-NCI and suggest that some dendritic injury in this condition is reversible.

EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment.

Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication.

N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of the Glutamine Antagonist 6-Diazo-5-oxo-l-norleucine (DON) as a Potential Treatment for HIV Associated Neurocognitive Disorders.

Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.

HIV infection model of chronic obstructive pulmonary disease in mice.

Cigarette smoke usage is prevalent in human immunodeficiency virus (HIV)-positive patients, and, despite highly active antiretroviral therapy, these individuals develop an accelerated form of chronic obstructive pulmonary disease (COPD). Studies investigating the mechanisms of COPD development in HIV have been limited by the lack of suitable mouse models. Here we describe a model of HIV-induced COPD in wild-type mice using EcoHIV, a chimeric HIV capable of establishing chronic infection in immunocompetent mice. A/J mice were infected with EcoHIV and subjected to whole body cigarette smoke exposure. EcoHIV was detected in alveolar macrophages of mice. Compared with uninfected mice, concomitant EcoHIV infection significantly reduced forced expiratory flow 50%/forced vital capacity and enhanced distal airspace enlargement following cigarette smoke exposure. Lung IL-6, granulocyte-macrophage colony-stimulating factor, neutrophil elastase, cathepsin G, and matrix metalloproteinase-9 expression was significantly enhanced in smoke-exposed EcoHIV-infected mice. These changes coincided with enhanced IκBα, ERK1/2, p38, and STAT3 phosphorylation and lung cell apoptosis. Thus, the EcoHIV smoke exposure mouse model reproduces several of the pathophysiological features of HIV-related COPD in humans, indicating that this murine model can be used to determine key parameters of HIV-related COPD and to test future therapies for this disorder.

Enhanced human immunodeficiency virus Type 1 expression and neuropathogenesis in knockout mice lacking Type I interferon responses.

The roles of Type I interferon (IFN) in human immunodeficiency virus Type 1 (HIV-1) neuropathogenesis are poorly understood; both protective and deleterious effects of IFN signaling have been described. We used genetically modified mice deficient in the Type I IFN receptor (IFNRKO) to analyze the progress of HIV-1 brain infection and neuropathogenesis in the absence of IFN signaling. IFNRKO and wild-type (WT) mice on the 129xSv/Ev or C57BL/6 strain backgrounds were infected systemically with EcoHIV, a chimeric HIV-1 that productively infects mice. IFNRKO mice showed higher HIV-1 expression in spleen and peritoneal macrophages and greater virus infiltration into the brain compared to WT mice. Neuropathogenesis was studied by histopathological, immunohistochemical, immunofluorescence, and polymerase chain reaction analyses of brain tissues after the virus was inoculated into the brain by stereotaxic intracerebral injection. Both IFNRKO and WT mice showed readily detectable HIV-1 and brain lesions, including microglial activation, astrocytosis, and increased expression of genes coding for inflammatory cytokines and chemokines typical of human HIV-1 brain disease. Parameters of HIV-1 neuropathogenesis, including HIV-1 expression in microglia/macrophages, were significantly greater in IFNRKO than in WT mice. Our results show unequivocally that Type I IFN signaling and responses limit HIV-1 infection and pathogenesis in the brains of mice.

Transmission of chimeric HIV by mating in conventional mice: prevention by pre-exposure antiretroviral therapy and reduced susceptibility during estrus.

Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV) cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1-7 nights. At 1-6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR) amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70-100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2',3'-dideoxcytidine (ddC) or tenofovir largely prevented their EcoHIV/NDK infection by mating (P<0.05 and P<0.003, respectively). In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003) but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.

Mice chronically infected with chimeric HIV resist peripheral and brain superinfection: a model of protective immunity to HIV.

Infection by some viruses induces immunity to reinfection, providing a means to identify protective epitopes. To investigate resistance to reinfection in an animal model of HIV disease and its control, we employed infection of mice with chimeric HIV, EcoHIV. When immunocompetent mice were infected by intraperitoneal (IP) injection of EcoHIV, they resisted subsequent secondary infection by IP injection, consistent with a systemic antiviral immune response. To investigate the potential role of these responses in restricting neurotropic HIV infection, we established a protocol for efficient EcoHIV expression in the brain following intracranial (IC) inoculation of virus. When mice were inoculated by IP injection and secondarily by IC injection, they also controlled EcoHIV replication in the brain. To investigate their role in EcoHIV antiviral responses, CD8+ T lymphocytes were isolated from spleens of EcoHIV infected and uninfected mice and adoptively transferred to isogenic recipients. Recipients of EcoHIV primed CD8+ cells resisted subsequent EcoHIV infection compared to recipients of cells from uninfected donors. CD8+ spleen cells from EcoHIV-infected mice also mounted modest but significant interferon-γ responses to two HIV Gag peptide pools. These findings suggest EcoHIV-infected mice may serve as a useful system to investigate the induction of anti-HIV protective immunity for eventual translation to human beings.

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370.

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.

Pathomorphologic and immunohistochemical study on the devastation of rat embryos by antiphospholipid antibody positive serum.

PROBLEM: While relying on previous publications, our aim was to examine the morphologic changes, induced in early rat embryos by intra-uterine exposure to the low-molecular weight fraction of boiled human serum containing antiphospholipid antibodies (APLA) that had been obtained from women with antiphospholipid syndrome (APS). METHOD OF STUDY: Human APLA-positive sera were pooled, boiled, centrifuged and separated by ultrafiltration. The molecular weight fraction lower than 30 kDa was used for the experiments. One hundred and fifty microlitres was injected into one uterine horn of 12 pregnant rats, 5 or 6 days after fertilization, while similarly prepared normal human serum or saline were injected into the contralateral horn. The rats were subsequently sacrificed. Serial sections, obtained from all uterine horns, were stained histologically and immunohistochemically. Normal embryos developed in the control uterine horns, while embryos in the experimental horns were destroyed rapidly. RESULTS: Signs of apoptosis appeared 2 hr following the injection, and 4 hr later all the embryonic cells were apoptotically destroyed. There was only partial damage to cytotrophoblasts and intermediate trophoblasts. CONCLUSION: These findings support the existence of a novel factor in the APLA-positive serum, causing a detrimental effect to the conceptus, without any relation to the antiphospholipid antibodies.

Oxygen consumption by a coral reef sponge.

Oxygen consumption of the Red Sea coral reef sponge Negombata magnifica was measured using both incubation and steady-state methods. The latter method was found to be the more reliable because sponge activity remained stable over time. Oxygen consumption rate was measured during three levels of sponge activity: full activity, reduced activity and basal activity (starved). It was found that the active oxygen consumption rate of N. magnifica averaged 37.3+/-4.6 nmol O2 min(-1) g(-1) wet mass, which is within the upper range reported for other tropical marine sponges. Fully active N. magnifica individuals consumed an average of 41.8+/-3.2 nmol O2 min(-1) g(-1) wet mass. The mean basal respiration rate was 20.2+/-1.2 nmol O2 min(-1) g(-1) wet mass, which is 51.6+/-2.5% of the active respiration rate. Therefore, the oxygen used for water pumping was calculated to be at most 10.6+/-1.8 nmol O2 min(-1) g(-1) wet mass, which is 25.1+/-3.6% of the total respiration. Combined oxygen used for maintenance and water pumping activity was calculated to be 30.8 nmol O2 min(-1) g(-1) wet mass, which is approximately 74% of the sponge's total oxygen requirement. The remaining oxygen is directed to other physiological activities, mainly the energy requirement of growth. These findings suggest that only a relatively minor amount of energy is potentially available for growth, and thus might be a factor in controlling the growth rate of N. magnifica in oligotrophic coral reefs.

Vaccine-induced protection from infection of mice by chimeric human immunodeficiency virus type 1, EcoHIV/NL4-3.

EcoHIV/NL4-3 is a chimeric human immunodeficiency virus type 1 (HIV-1) that can productively infect mice. This study tests the utility of EcoHIV/NL4-3 infection to reveal protective immune responses to an HIV-1 vaccine. Immunocompetent mice were first immunized with VRC 4306 which encodes subtype B consensus sequences of gag, pol, and nef and then were infected by EcoHIV/NL4-3. Anti-Gag antibodies were sampled during immunization and infection. The extent of EcoHIV/NL4-3 infection in spleen cells and peritoneal macrophages was determined by quantitative real-time PCR (QPCR). Although antibody titres were not significantly different in control and vaccinated groups, VRC 4306 immunization induced protective responses that significantly reduced virus burden in both lymphocyte and macrophage compartments. These results indicate that EcoHIV/NL4-3 infection can be controlled by HIV-1 vaccine-induced responses, introducing a small animal model to test vaccine efficacy against HIV-1 infection.

Testing antiretroviral drug efficacy in conventional mice infected with chimeric HIV-1.

OBJECTIVE: We previously described chimeric HIV-1, EcoHIV, which can infect mouse cells in culture and cause spreading infection in conventional immunocompetant mice. We have now applied this system as a model for preclinical evaluation of anti-retroviral drugs. DESIGN AND METHODS: We used chimeric virus EcoHIV/NDK constructed on the backbone of subtype D NDK. EcoHIV/NDK expression in mice was characterized 5-10 days after infection by testing viral DNA, RNA, and protein burdens in spleen and macrophages by real-time PCR (QPCR), RT-PCR, and p24 ELISA. For antiviral evaluation, groups of 5-7 mice were pretreated with 2',3'-dideoxycytidine (ddC), abacavir, or vehicle; mice were then infected with EcoHIV/NDK, treatment maintained for additional 48 h, and tested for viral DNA and RNA burdens in spleens and macrophages by QPCR. RESULTS: EcoHIV/NDK infected mice reproducibly showed viral burdens of up to 1.4 x 10 viral DNA copies and 200 pg p24 per 10 spleen cells and expressed spliced Vif RNA and mature p24 in macrophages 5-10 days after infection. Treatment of mice with 60 or 300 mg ddC/kg/day blocked EcoHIV/NDK infection in a dose-dependent manner with significantly lower viral DNA and RNA burdens at both drug doses (P < 0.001) in the spleens of infected mice. Abacavir tested at 100 mg/kg/day caused 96% inhibition of viral DNA synthesis in spleen and it almost completely abolished viral spliced RNA synthesis in spleens and macrophages. CONCLUSIONS: The system of chimeric HIV-1 infection of mice permits rapid, statistically powerful, and inexpensive evaluation of antiretroviral drugs in vivo.

Effect of intrauterine injection of casein on fetal survival in rat: a new pharmacological approach for contraception.

BACKGROUND: The incidental finding of casein as a possible new local pharmacological contraceptive prompted us to assess its validity in an experimental rat model. METHODS: The intrauterine injection of 150 microg of bovine alpha-casein dissolved in 150 microL phosphate-buffered saline (PBS) was performed on one uterine rat horn on days L(5)-L(7), whereas the contralateral horn was used for injection of 150 microL PBS as a control. Intraperitoneal injection of alpha-casein (5 mg/mL, 2 mL/rat) was performed on day L(5). The rats were killed by cervical dislocation on the day L(14). RESULTS: The effect of an alpha-casein on fetal resorption rate was assessed following the unilateral injection of 150 microL of alpha-casein (1 mg/mL in PBS) and compared with the effect obtained following the contralateral injection of 150 microL PBS. The unilateral injection of alpha-casein on day L(5) caused a significant increase in fetal resorption rate as compared with the contralateral uterine horn injected with PBS (p<.00001). The decrease in alpha-casein concentration from 1 to 0.3 mg/mL caused a reduced, but still significant, effect on fetal resorption rate (p<.0001). The injection on days L(6)-L(7) caused a local effect of resorption near the injection site. There was no effect on fetal resorption rate following the injection of alpha-casein intraperitoneally. CONCLUSION: Our data suggest a new pharmacological approach for contraception, based on local intrauterine effect of alpha-casein in an experimental rat model. The exact mechanism of action related to casein should be further studied.