No evidence for enhanced disease with human polyclonal SARS-CoV-2 antibody in the ferret model.

Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-dependent enhancement (ADE) of disease as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral/ocular) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7-10 days post-infection. To evaluate the potential for ADE, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.

Human immunoglobulin from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike antigen efficiently neutralizes viral variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.

In Vitro and In Vivo Phenotypes of Venezuelan, Eastern and Western Equine Encephalitis Viruses Derived from cDNA Clones of Human Isolates.

The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA "Animal Rule" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other "wild type" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

Human immunoglobulin from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike antigen efficiently neutralizes viral variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.

LDLRAD3 is a receptor for Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans1. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.

Doping of ZnO inorganic-organic nanohybrids with metal elements.

We present a general and in-depth study of the effect of dopants in hybrid inorganic/organic ZnO/PAA (polyacrylic acid) nanocomposites. These dopants vary as much by their ionic size, as by their electronic valence and some of them have been used in ZnO due to their known magnetic and/or optical properties. The chemical nature of the dopants controls their ability to incorporate into ZnO crystal lattice. Three concentrations (0.1%, 1% and 5%) of dopants were studied in order to compare the effect of the concentration with the results obtained previously in the literature. Our results confirm in the first place the trend observed in the literature, that increase in dopant concentration leads to quenching of visible luminescence for ZnO nanocrystals obtained by very different processes. However, the degradation of photoluminescence quantum yield (PL QY) is not inevitable in our nanocomposites. At low doping concentration for some dopants with a small or comparable ionic radius than Zn2+, PL QY can be maintained or even improved, making it possible to tune the visible emission spectrum between 2.17 eV and 2.46 eV. This opens up the prospect of synthesizing phosphors without rare earth for white LEDs, whose spectrum can be tuned to render warm or cold white light, by a chemical synthesis process with a low environmental impact.

The Use of Phage Display and Yeast Based Expression System for the Development of a Von Willebrand Factor Propeptide Assay: Development of a Von Willebrand Factor Propeptide Assay.

BACKGROUND: The diagnosis of von Willebrand disease is complex due to the heterogeneity of the disease. About eighty percent of von Willebrand disease patients are diagnosed with a quantitative defect of von Willebrand factor (VWF) where fifty percent is due to an increased clearance of von Willebrand factor. These patients do not respond well to the treatment of choice, Desmopressin (DDAVP) due to decreased efficacy. The ratio between the VWF propeptide and the mature VWF antigen is used to diagnose these patients. Commercial VWF propeptide assays are too expensive for use in developing countries. In this study, we developed a cost-effective ELISA assay. METHODS: We first displayed VWF propeptide on yeast. Antibody fragments were selected against the displayed VWF propeptide by using phage display technology. The antibodies were used to develop a cost-effective VWF propeptide assay and compared to a commercial VWF propeptide assay. RESULTS: Two of these antibody fragments bound specific to the VWF propeptide and not to the yeast used for the expression of the propeptides. These purified antibody fragments were able to detect VWF propeptide in normal plasma. CONCLUSION: Our assay performed well when compared to a commercial kit. It also showed a higher binding affinity for VWF propeptide in plasma at especially lower plasma concentrations.

Evaluation of nucleic acid stabilization products for ambient temperature shipping and storage of viral RNA and antibody in a dried whole blood format.

Loss of sample integrity during specimen transport can lead to false-negative diagnostic results. In an effort to improve upon the status quo, we used dengue as a model RNA virus to evaluate the stabilization of RNA and antibodies in three commercially available sample stabilization products: Whatman FTA Micro Cards (GE Healthcare Life Sciences, Pittsburgh, PA), DNAstable Blood tubes (Biomatrica, San Diego, CA), and ViveST tubes (ViveBio, Alpharetta, GA). Both contrived and clinical dengue-positive specimens were stored on these products at ambient temperature or 37°C for up to 1 month. Antibody and viral RNA levels were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays, respectively, and compared with frozen unloaded controls. We observed reduced RNA and antibody levels between stabilized contrived samples and frozen controls at our earliest time point, and this was particularly pronounced for the FTA cards. However, despite some time and temperature dependent loss, a 94.6-97.3% agreement was observed between stabilized clinical specimens and their frozen controls for all products. Additional considerations such as cost, sample volume, matrix, and ease of use should inform any decision to incorporate sample stabilization products into a diagnostic testing workflow. We conclude that DNAstable Blood and ViveST tubes are useful alternatives to traditional filter paper for ambient temperature shipment of clinical specimens for downstream molecular and serological testing.

Immune response to and histopathology of Campylobacter jejuni infection in ferrets (Mustela putorius furo).

Campylobacter jejuni is 1 of the most common enteric bacterial pathogens worldwide. The mechanisms of pathogenesis remain obscure, in part because of limitations of small animal models. Young ferrets develop diarrhea when fed C. jejuni, but their pathology and the immune response after infection have not been examined in detail. In the present study, we examined the pathogenesis of C. jejuni CG8421 and associated immune responses in ferrets. After oral infection with C. jejuni CG8421, 86.7% of the animals developed diarrhea and inflammatory responses that were similar to those seen in human infection. Pronounced histopathologic changes in the colonic mucosa of infected animals were observed during the acute phase (days 1 through 3) of infection. Electron micrographs of colonic epithelium revealed disruption of the villi and internalized bacteria that were not within membrane vacuoles. During the acute phase, C. jejuni was isolated from the livers of 7 of 9 (78%) animals, and bacteria were visualized immunohistochemically in the livers from 5 of the 7 animals (71%) from which C. jejuni was isolated. A vigorous systemic and mucosal immune response to Campylobacter antigens was elicited after infection of ferrets. The data presented contribute to the current knowledge of the pathogenicity of and immunologic response to C. jejuni CG8421 in ferrets and better understanding of this model.

Body mass index and the risk of postoperative complications with dentoalveolar surgery: a prospective study.

OBJECTIVES: Obesity is a disease that affects millions of Americans. Sixty-nine million Americans are considered to be overweight including at least 1 in 5 children. Overweight and obesity as risk factors for complications associated with dentoalveolar surgery have not been extensively studied. The purpose of this study was to prospectively investigate the frequency and the nature of postoperative complications in obese patients receiving ambulatory dentoalveolar surgery. STUDY DESIGN: Patients undergoing dentoalveolar outpatient ambulatory surgery at the University of Cincinnati Oral and Maxillofacial Surgery Clinic from January 10, 2005 to December 7, 2005 were enrolled in a prospective study. At the preoperative visit, height, weight, and age were recorded. The body mass index (BMI) was calculated and patients were divided into weight categories based on CDC and WHO classifications. Unfavorable outcomes studied included number of postoperative visits, infections, alveolar osteitis, soft and hard tissue problems, and postoperative bleeding. Complication categories were created for all complications identified during postoperative exams and t tests were performed to see if increased BMI correlated to higher postoperative complication rates, if multiple complications correlated to increased BMI, and to see if individual postoperative complication groups and BMI correlated. Because it was thought that complications would increase the number of postoperative exams for a particular patient, simple linear regression was used to see if BMI correlated to increased number of postoperative visits. RESULTS: No significant differences were noted in number of postoperative complications when comparing patients' BMIs (t = -0.62, P = .5350). Simple linear regression analysis showed that increased BMI did not predict increased number of postoperative visits. Increased age was the only predictor of increased postoperative complication risk (t = -3.17, P = .0016) and of increased number of postoperative visits (t = -16.35, P < .0001, and P < .0001 by nonparametric Wilcoxon rank sum test). CONCLUSIONS: The BMI is not statistically correlated to higher postoperative complication rates or increased number of postoperative visits. Obesity remains an important risk factor for dentoalveolar ambulatory outpatient anesthesia; however, it is not associated with poorer postoperative complication rates.

Immunogenicity and protective efficacy of recombinant Campylobacter jejuni flagellum-secreted proteins in mice.

Immunogenicity and protective efficacy of three Campylobacter jejuni flagellum-secreted proteins, FlaC, FspA1, and FspA2, were compared by use of a mouse model. Mice were immunized intranasally with each protein with or without LTR192G as the adjuvant and challenged intranasally with C. jejuni 81-176 or CG8486. All three proteins were immunogenic, although FspA1 induced the highest levels of serum immunoglobulin G (IgG) and fecal IgA. Although immunogenic, FlaC provided only 18% protection against disease from C. jejuni 81-176. Immunization with FspA1 resulted in 57.8% protection without adjuvant or 63.8% protection with adjuvant against homologous challenge with 81-176. Alternatively, immunization with FspA2 provided 38.4% (without adjuvant) or 47.2% (with adjuvant) protection against disease from homologous challenge with CG8486. In contrast to FspA2, FspA1 provided some heterologous protection against C. jejuni CG8486 when delivered with (31.2%) or without (44.8%) LTR192G. These results suggest that FspA1 may be a good subunit vaccine candidate against C. jejuni disease.

Towards the world-wide ban of indoor cigarette smoking in public places.

In 1984, Dr. C. Everett Koop, then Surgeon General of the US, presented an important speech on the hazards of smoking. In his speech, he stated "The ultimate goal should be a smoke-free society by the year 2000." In addition, the World Health Organization (WHO) has initiated a process to ban smoking globally; on 21 May 2003, at the 56th World Health Assembly, WHO's 192 Member States unanimously adopted the world's first public health treaty, the WHO Framework Convention on Tobacco Control. Although good progress has been made, reaching the ultimate goal is far from certainty. Therefore, it is time to re-visit this crucial public health activity and re-energize the effort to reach this goal. Since numerous reports have been written on the ban of smoking based on benefits to the smokers, the emphasis of our report is on benefits to non-smokers from their exposure to environmental tobacco smoke (ETS). We provide a concise review of the impact of ETS on health and economy. In addition, we examined the different interest groups on supporting and opposing the ban, the role of the government, private citizens and medical professionals on this activity, and certain constraints on implementing the global ban. We also provide some recommendations on how to promote the ban globally. Since cigarette smoking is an unnecessary habit that has devastating consequences around the world, banning of cigarette smoking should be a global mission. A global ban on indoor smoking in public places is an important first step in an international effort to prevent morbidity and mortality caused by tobacco smoking and ETS.

Life threatening massive pulmonary embolism treated with reteplase: a case report.

We describe a case of sudden and severe pulseless electrical activity in a 30 year old woman which was managed successfully with reteplase and heparin one day following an anterior cruciate ligament repair. The presentation of a sudden collapse with ECG findings of S1Q3T3, early precordial lead ST depression and partial right bundle branch block were indicative of an acute pulmonary embolus. The cardiopulmonary collapse necessitated rapid treatment in the absence of confirmatory investigations. Reteplase (10 U stat followed by 10 U at 30 minutes) led to a dramatic improvement in the cardiovascular status of the patient. One day following the cardiac arrest the patient was extubated and responding normally. A spiral CT performed later confirmed multiple small embolic defects in the lower pulmonary arteries of both lower lung zones. This case highlights the utility of reteplase in the management of an acute pulmonary embolism and in an emergency, recent surgery is not necessarily a contraindication to its use.

Effects of DSP-4 on monoamine and monoamine metabolite levels and on beta adrenoceptor binding kinetics in rat brain at different times after administration.

Effects of DSP-4 on noradrenaline (NA), 3-methoxy-4-hydroxyphenyl glycol (MHPG), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) levels and on beta adrenoceptor binding kinetics (Bmax and KD) in rat hippocampus, cortex and hypothalamus were studied between 24 hours and 14 days after systemic administration. Beta adrenoceptor numbers in hippocampus and cortex, but not in hypothalamus, were significantly increased after DSP-4. No significant changes in KD values were observed in hypothalamus, but significant increases in this parameter were measured in hippocampus and cortex. NA and MHPG levels were significantly decreased in all three brain regions, but MHPG/NA ratios were increased in hippocampus, decreased in cortex and unchanged in hypothalamus. Very prominent increases in 5-HIAA levels were observed in all three brain regions, but only at one day after DSP-4. The greatest increases in 5-HIAA levels occurred in the hippocampus, but this effect of DSP-4 appeared to be slightly diminished by pre-treatment with fluoxetine. In cortex and hippocampus 5-HT levels were slightly, but significantly decreased after DSP-4.

Estrogen-2/4-hydroxylase activities in rat brain and liver microsomes exhibit different substrate preferences and sensitivities to inhibition.

NADPH-dependent estrogen-2/4-hydroxylase activities in rat brain and liver microsomes were compared with respect to the utilization of different estrogens as substrates and the inhibitory effects of alpha-naphthoflavone, metyrapone and steroids. Of 6 different estrogens used as substrates, only 17 beta- and 17 alpha-estradiol were transformed relatively effectively by brain microsomes. In contrast liver microsomes utilized these two estrogens as well as ethynyl estradiol, estrone and diethylstilbestrol effectively. Estriol was a poor substrate for estrogen-2/4-hydroxylase activity in both tissues. With 40 microM 17 beta-estradiol as substrate the estrogen-2/4-hydroxylase activities in brain and liver were inhibited by alpha-naphthoflavone, metyrapone, progesterone, 17 alpha-hydroxyprogesterone and testosterone. The brain enzyme activity appeared to be more sensitive than the liver enzyme to inhibition by alpha-naphthoflavone and metyrapone. Testosterone propionate (50-100 microM) stimulated the brain enzyme activity significantly. Progesterone and 17 alpha-hydroxyprogesterone were the most effective steroidal inhibitors of brain estrogen-2/4-hydroxylase activity. In the liver the inhibitory potencies of 3 different steroids varied, depending on the estrogen used as substrate. With 17 beta-estradiol, for example, progesterone was the most potent steroidal inhibitor, while corticosterone was the most potent inhibitor when diethylstilbestrol was used as substrate. These findings indicate that rat liver microsomes can utilize a wider range of different estrogens for catecholestrogen formation than brain microsomes and suggest that the profiles of catecholestrogen-forming P-450 isozymes in the two organs differ.

Substrate dependency of specific and non-specific estrogen-2/4-hydroxylase activities measured by the radio-enzymatic method in rat brain microsomes.

Putative specific and non-specific estrogen-2/4-hydroxylase activities which might affect the radio-enzymatic assay were characterized in terms of their requirements for NADPH and their substrate dependency. Using rat brain microsomes and partially purified rat liver COMT three main sources of estrogen-2/4-hydroxylase activity could be distinguished; a COMT-related component and NADPH-dependent and NADPH-independent microsomal components. The COMT-related activity required NADPH and showed about equal preferences for estrone and estradiol. The NADPH-dependent component was highly specific for estradiol, the relative activities observed with estrone and estriol being 7 and 1% of that observed with estradiol. The NADPH-independent component exhibited substrate saturation, was heat-labile and could not be inhibited by alpha-naphthoflavone or metyrapone. It showed a preference for estrone over estradiol, with estriol being a very poor substrate. These findings indicate that non-enzymatic factors contribute very little to product formation in the radio-enzymatic assay. The specificity of the major NADPH-dependent microsomal component towards estradiol suggests a stereo-specific requirement for the D-ring configuration of this estrogen. The use of no-cofactor blanks in the radio-enzymatic assay may be very important when different estrogens are compared as substrates for estrogen-2/4-hydroxylases.

Evidence that estradiol-2/4-hydroxylase activities in rat hypothalamus and hippocampus differ qualitatively and involve multiple forms of P-450: ontogenetic and inhibition studies.

Estradiol-2/4-hydroxylase activity was studied in hypothalamic and hippocampal microsomes of male and female Wistar rats between 22 and 75 days after birth. The activity exhibited substrate saturation (50-100 microM estradiol) required NADPH and was inhibited by carbon monoxide, alpha-naphthoflavone and metyrapone. With 30 microM estradiol, 50% inhibition required 50-70 microM alpha-naphthoflavone compared to 200 microM metyrapone. Metyrapone exhibited biphasic inhibition curves which did not differ significantly between hypothalamus and hippocampus, whereas alpha-naphthoflavone was a more potent inhibitor of the hippocampal enzyme than of the hypothalamic enzyme. The Km and Vmax of the hippocampal estradiol-2/4-hydroxylase were significantly greater than that of the hypothalamus in both sexes at all ages studied. In female rats the Km of the hypothalamic enzyme changed from 14 microM at 23 days of age, to 47 microM at 71 days, but remained constant at about 29 microM in males. The Km of the hippocampal enzyme showed no significant change with age in either sex. The present findings indicate that catechol estrogen formation in the brain is catalyzed by multiple forms of microsomal P-450. They also suggest that these enzyme activities in the rat hypothalamus and hippocampus differ qualitatively. Ontogenetic changes in the Km of estradiol-2/4-hydroxylases appeared to be limited to the female hypothalamus. This might reflect a specific biological requirement of the female hypothalamus during critical stages of sexual differentiation of the brain. The relatively high hippocampal activity might reflect the catalytic versatility of different P-450 isozymes and does not necessarily imply a physiologically meaningful role with respect to catechol estrogen biosynthesis in this particular brain area.

Ontogeny of estradiol-2-hydroxylase activity in discrete brain areas of Wistar rats.

Estradiol-2-hydroxylase activity was assayed radio-enzymatically in microsomes of six different brain areas of male and female Wistar rats between 22 and 77 days of age. The hypothalamus consistently showed the lowest activity and the hippocampus the highest, with the other areas falling in between. This pattern was seen in both sexes and at all the ages studied. In both sexes all six brain areas showed a steady increase in activity with increasing age. No statistically significant sexual differences in activity were observed for any area. The relatively low estradiol-2-hydroxylase activity in the hypothalamus is noteworthy since it contradicts the published evidence that this area has the highest in vitro catecholestrogen forming capacity in the brain.