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Efficient lysis is critical when analyzing single cells in microfluidic droplets, but existing methods utilize detergents that can interfere with the assays to be performed. We demonstrate robust cell lysis without the use of detergents or other chemicals. In our method, cells are exposed to electric field immediately before encapsulation in droplets, resulting in cell lysis. We characterize lysis efficiency as a function of control parameters and demonstrate compatibility with enzymatic assays by measuring the catalysis of β-glucosidase, an important cellulase used in the conversion of biomass to biofuel. Our method enables assays in microfluidic droplets that are incompatible with detergents.

The Plasmodium vivax Merozoite Surface Protein-3α (PvMSP-3α) is considered as a potential vaccine candidate. However, the detailed investigations of the type of immune responses induced in naturally exposed populations are necessary. Therefore, we aim to characterize the naturally induced antibody to PvMSP-3α in 282 individuals with different levels of exposure to malaria infections residents in Brazilian Amazon. PvMSP3 specific antibodies (IgA, IgG and IgG subclass) to five recombinant proteins and the epitope mapping by Spot-synthesis technique to full-protein sequence of amino acids (15aa sequence with overlapping sequence of 9aa) were performed. Our results indicates that PvMSP3 is highly immunogenic in naturally exposed populations, where 78% of studied individuals present IgG immune response against the full-length recombinant protein (PVMSP3-FL) and IgG subclass profile was similar to all five recombinant proteins studied with a high predominance of IgG1 and IgG3. We also observe that IgG and subclass levels against PvMSP3 are associated with malaria exposure. The PvMSP3 epitope mapping by Spot-synthesis shows a natural recognition of at least 15 antigenic determinants, located mainly in the two blocks of repeats, confirming the high immunogenicity of this region. In conclusion, PvMSP-3α is immunogenic in naturally exposed individuals to malaria infections and that antibodies to PvMSP3 are induced to several B cell epitopes. The presence of PvMSP3 cytophilic antibodies (IgG1 and IgG3), suggests that this mechanism could also occur in P. vivax., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Plasmodium vivax merozoite surface protein (PvMSP9) stimulates both cellular and humoral immune responses in individuals who are naturally infected by this parasite species. To identify immunodominant human T-cell epitopes in PvMSP9, we used the MHC class II binding peptide prediction algorithm ProPred. Eleven synthetic peptides representing predicted putative promiscuous T-cell epitopes were tested in IFN-gamma and IL-4 ELISPOT assays using peripheral blood mononuclear cells (PBMC) derived from 142 individuals from Rondonia State, Brazil who had been naturally exposed to P. vivax infections. To determine whether the predicted epitopes are preferentially recognized in the context of multiple alleles, MHC Class II typing of the cohort was also performed. Five synthetic peptides elicited robust cellular responses, and the overall frequencies of IFN-gamma and IL-4 responders to at least one of the promiscuous peptides were 62% and 46%, respectively. The frequencies of IFN-gamma and IL-4 responders to each peptide were not associated with a particular HLA-DRB1 allelic group since most of the peptides induced a response in individuals of 12 out of 13 studied allelic groups. The prediction of promiscuous epitopes using ProPred led to the identification of immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous population exposed to malaria infections. The combination of several such T-cell epitopes in a vaccine construct may increase the frequency of responders and the overall efficacy of subunit vaccines in genetically distinct populations., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The theoretical study of plasma turbulence is of central importance to fusion research. Experimental evidence indicates that the confinement time results mainly from the turbulent transport of energy, the magnitude of which depends on the turbulent state resulting from nonlinear saturation mechanisms, in particular, the self-generation of coherent macroscopic structures and large scale flows. Plasma geometry has a strong impact on the structure and magnitude of these flows and also modifies the mode linear growth rates. Nonlinear global gyrokinetic simulations in realistic tokamak magnetohydrodynamic equilibria show how plasma shape can control the turbulent transport. Results are best described in terms of an effective temperature gradient. With increasing plasma elongation, the nonlinear critical effective gradient is not modified while the stiffness of transport is decreasing.

Background: The transversus abdominis plane (TAP) block is a new regional anaesthesia technique that provides analgesia after abdominal surgery. It involves injection of local anaesthetic into the plane between the transversus abdominis and the internal oblique muscles. The TAP block can be performed using a landmark technique through the lumbar triangle or with ultrasound guidance. The goal of this anatomical study with dye injection into the TAP and subsequent cadaver dissections was to establish the likely spread of local anaesthesia in vivo and the segmental nerve involvement resulting from ultrasound-guided TAP block., Methods: An ultrasound-guided injection of aniline dye into the TAP was performed for each hemi-abdominal wall of 10 unembalmed human cadavers and this was followed by dissection to determine the extent of dye spread and nerve involvement in the dye injection., Results: After excluding one pilot specimen and one with advanced tissue decomposition, 16 hemi-abdominal walls were successfully injected and dissected. The lower thoracic nerves (T10-T12) and first lumbar nerve (L1) were found emerging from posterior to anterior between the costal margin and the iliac crest. Segmental nerves T10, T11, T12, and L1 were involved in the dye in 50%, 100%, 100%, and 93% of cases, respectively., Conclusions: This anatomical study shows that an ultrasound-guided TAP injection cephalad to the iliac crest is likely to involve the T10-L1 nerve roots, and implies that the technique may be limited to use in lower abdominal surgery.

Antibody and T-cell reactivities to Plasmodium vivax merozoite surface protein 9 (PvMSP9) were evaluated in a cross-sectional study of individuals naturally exposed to malaria infections living in Ribeirinha, a native riverine community and in Colina, a transmigrant community, Rondonia, Brazil. The antibody responses to PvMSP9-RIRIIand PvMSP9-Nt domains in Ribeirinha were higher compared with Colina and correlated with age and time of malaria exposure. IgG2 was most prevalent for PvMSP9-RII in both communities, and IgG1 was the predominant isotype for PvMSP9-Nt and PvMSP9-RIRII in Ribeirinha. IFN-gamma and IL-4 predominated in Ribeirinha, while IFN-gamma predominated in Colina. Variation in exposure to P. vivax likely accounts for the differences observed in cytokine and antibody levels between the two populations studied.

Previous descriptions of the thoracolumbar spinal nerves innervating the anterior abdominal wall have been inconsistent. With modern surgical and anesthetic techniques that involve or may damage these nerves, an improved understanding of the precise course and variability of this anatomy has become increasingly important. The course of the nerves of the anterior abdominal is described based on a thorough cadaveric study and review of the literature. Twenty human cadaveric hemi-abdominal walls were dissected to map the course of the nerves of the anterior abdominal wall. Dissection included a comprehensive tracing of nerves and their branches from their origins in five specimens. The branching pattern and course of all nerves identified were described. All thoracolumbar nerves that innervate the anterior abdominal wall were found to travel as multiple mixed segmental nerves, which branch and communicate widely within the transversus abdominis plane (TAP). This communication may occur at multiple locations, including large branch communications anterolaterally (intercostal plexus), and in plexuses that run with the deep circumflex iliac artery (DCIA) (TAP plexus) and the deep inferior epigastric artery (DIEA) (rectus sheath plexus). Rectus abdominis muscle is innervated by segments T6-L1, with a constant branch from L1. The umbilicus is always innervated by a branch of T10. As such, identification or damage to individual nerves in the TAP or within rectus sheath is unlikely to involve single segmental nerves. An understanding of this anatomy may contribute to explaining clinical outcomes and preventing complications, following TAP blocks for anesthesia and DIEA perforator flaps for breast reconstruction., ((c) 2008 Wiley-Liss, Inc.)

There is a wide range of indications for therapeutic bronchoscopy in children today: foreign body removal, bronchoaspiration, endoscopy-assisted tracheal intubation, selective intubation and airway management during thoracic surgery or in children undergoing mechanical ventilation. Some adult-derived methods may find potential indications in pediatric patients: airway stenosis dilation, laser photoresection, tracheobronchial stenting. There are no rules regarding such procedures in children, and supposed benefits have to be weighted against those of more conventional therapies.

We have identified a third member of the junctional adhesion molecule (JAM) family. At the protein level JAM3 displays 36 and 32% identity to JAM2 and JAM1, respectively. The coding region is distributed over 9 exons and maps to chromosome 11q25. The gene shows widespread tissue expression with higher levels apparent in the kidney, brain, and placenta. At the cellular level we show expression of JAM3 transcript within endothelial cells. Our major finding is that JAM3 and JAM2 are binding partners. Thus, JAM3 ectodomain binds firmly to JAM2-Fc. This heterotypic interaction is maintained when JAM3-Fc is used to capture Chinese hamster ovary cells expressing full-length JAM2. In static adhesion assays we show that JAM3 is unable to bind to leukocyte cell lines. This is consistent with the lack of JAM2 expression. However, using JAM2-Fc pull-down experiments in combination with polyclonal anti-JAM3 serum, we demonstrate that JAM3 is the previously uncharacterized 43-kDa counter-receptor that mediates JAM2 adhesion to T cells. Most significantly we demonstrate up-regulation of JAM3 protein on peripheral blood lymphocytes following activation. Finally we show the utility of JAM3 ectodomain as an inhibitor of JAM2 adhesion.

A broadly used pan-HLA class I-reactive monoclonal antibody W6/32 is believed to recognize a conformational epitope dependent on association between heavy chains and beta2-microglobulin (beta2m). However, in the present study we report that W6/32 does recognize at least some free HLA class I heavy chains under the partially denaturating conditions of nonreducing Western blotting, namely nearly all HLA-B allelic products. Furthermore, we confirm and largely extend our previous observation that complexes of beta2m with heavy chains of a few HLA class I allelic forms (most notably HLA-B27) exhibit unusual resistance to dissociation by SDS, which is reminiscent of MHC class II molecules. In addition, our data indicate the existence of covalent (disulfide-linked) heterodimers of certain HLA class I heavy chains (namely Cw1 and Cw4) and beta2m.

Objectives: The presence of low grade dysplasia (LGD) within Barrett's esophagus (BE) has a multitude of ramifications. Identification of markers that could risk stratify LGD would be of great clinical benefit. We aimed to prospectively evaluate the prognosis of the immunohistochemical overexpression of p53 protein in BE colocalized to LGD., Methods: Consecutive BE patients in whom LGD was found had a repeat esophagogastroduodenoscopy within 8-12 wk per an ongoing prospective study. At each esophagogastroduodenoscopy, a therapeutic scope was used in conjunction with the Seattle Biopsy Protocol. Patients were observed until development of multifocal high grade dysplasia (mHGD), presence of an HGD dysplasia-associated lesion or mass (DALM) lesion, or frank adenocarcinoma. p53 protein overexpression was determined by computerized immunoquantitation using image analysis software on step serial-sectioned specimens of BE segment(s) harboring LGD. Kaplan-Meier survival curves were made on the ability of p53 staining colocalized to areas of LGD to predict progression to mHGD, HGD DALM, or cancer during prospective follow-up., Results: Forty-eight BE patients with LGD were observed for a mean of 41.2+/-22.5 months. During this period, five of 48 patients progressed to mHGD with a focus in which intramucosal cancer could not be excluded (one), mHGD/DALM with one or more foci in which intramucosal cancer could not be excluded (two), cancer (one), or mHGD (one). Twelve had persistent LGD and 31 had regressed to no dysplasia. p53 staining was positive and colocalized to areas of LGD in 4/31 of patients that regressed, 3/12 that persisted, and 3/5 that progressed. Kaplan-Meier curves differed significantly between p53 positive and negative patients for outcome defined as progression of LGD., Conclusions: p53 colocalization with LGD at index LGD diagnosis is a risk factor for progression of LGD. This can potentially be used to risk stratify BE LGD patients in terms of surveillance intervals or enrollment into secondary prevention studies.

In Vietnam, information about blood pressure, serum lipids and their factors is limited. To obtain some of this information, a cross sectional nutrition survey was carried out in an urban and rural area of Ho Chi Minh City with 217 participants aged 60-69 y (148 females and 69 males). Anthropometry and blood pressure were measured. For three consecutive weekdays, 24 h dietary recalls were performed. Single 24 h urine was collected for sodium and potassium analysis. A fasting blood sample was taken and biochemical parameters were measured. Results indicate a high percentage of hypertension in urban (female: 35.5%, male: 43.8%) and rural areas (female: 22.2%, male: 35.1%). Blood pressure was correlated with body mass index (BMI) and 24 h urinary sodium-to-potassium (Na/K) ratio. A high prevalence of serum total cholesterol (TC) above 220 mg/dL (female: 55.3%, male: 31.3%) and overweight (female: 34.2%, male: 25.0%) were observed in urban residents. By contrast, 5.6% and 24.3% of rural females and males respectively had TC below 150 mg/dL and both genders had the same prevalence of underweight (32.4%). TC was positively correlated with body weight, BMI, dietary protein and dietary lipids. Overweight might be a major risk factor for hypertension in our urban elderly. A high Na/K intake ratio might be a risk factor for hypertension in both areas. The high prevalence of elevated TC in the urban area might to be related to the high lipid intake, and the high prevalence of low TC in the rural area might to be related to the low lipid intake.

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with beta(2)-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with beta(2)-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/beta(2)-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.

Objective: To determine whether the enhancement of threshold sural sensory nerve action potentials (SNAPs) occurred in patients with spinal cord injury after upper limb isometric contraction., Design: This prospective study, in which ten paraplegic patients with spinal cord injury were recruited from the Harris County community and served as his/her own control, was performed in the electromyography laboratory at Harris County Hospital District Quentin Mease Hospital. The baseline SNAP was established using ten threshold, signal-averaged stimuli to the sural nerve. With the same stimulus strength, the SNAP was recorded while the subjects' arms were pulled apart against a force gauge at 50% and 100% maximum force. Responses were recorded every 4 min until the values returned to baseline., Results: Results showed an increase in the SNAP amplitude after 50% and 100% maximum force. The mean preexercise SNAP amplitude was 4.0 +/- 0.6 (SE) microV. At 50% force, the SNAP amplitude was 7.57 +/- 1.2 microV; at 100% force, it was 7.29 +/- 1.2 microV. The post hoc analysis of the data revealed P = 0.009 and P = 0.01 for 50% and 100% maximum force, respectively., Conclusions: The threshold SNAP of the sural nerve was enhanced after isometric exercise in paraplegic patients with spinal cord injury.

We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR antibody is able to completely block VEGF165-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF165. Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca2+ in HUVEC in response to VEGF165.

In an open-label, randomized trial, 44 Vietnamese children with diphtheria were given penicillin therapy (intramuscular benzylpenicillin, 50,000 U/[kg.d] for 5 days and then oral penicillin, 50 mg/[kg.d] for 5 days), and 42 were given erythromycin therapy (50 mg/[kg.d] orally for 10 days). There were no differences in times to membrane clearance or bacteriologic clearance, but median times to fever clearance were 27 hours (95% confidence interval [CI], 19-30; range, 0-124 hours) for penicillin recipients and 46 hours (95% CI, 34-54; range, 0-148 hours) for erythromycin recipients (P = .0004). In the penicillin group, acute treatment failed for one patient, and one patient relapsed. Three patients in the penicillin group developed diphtheritic myocarditis as evidenced by abnormal electrocardiograms. Erythromycin did not cause prolongation of the QT interval corrected for heart rate. Cultures of specimens from 15 patients (17.4%) were positive for toxigenic Corynebacterium diphtheriae. All isolates were susceptible to penicillin, but for isolates (27%), all of which were from patients who received penicillin treatment, were resistant to erythromycin (minimum inhibitory concentrations, > 64 mg/L). Penicillin is recommended as first-line treatment for diphtheria in Vietnam.

Malabsorption of fat is an important gastrointestinal cause of malnutrition and growth retardation in childhood. The gold standard for the evaluation of fat malabsorption is the faecal fat balance method. The acid steatocrit method has recently been introduced as a simple method to evaluate faecal fat. The present study was aimed at evaluating the acid steatocrit in clinical practice. Faecal fat excretion and acid steatocrit results were determined in 42 children, half with and half without fat malabsorption. Acid steatocrit results correlated significantly with both faecal fat excretion (p < 0.01) and faecal fat concentration (p < 0.001). Sensitivity and specificity of the acid steatocrit for the diagnosis of malabsorption were 90% and 100%, respectively. We consider the acid steatocrit method useful for the screening and monitoring of patients with steatorrhoea.

We describe a 45-year-old man who presented with multiple nodules along the tendons of the scapular region, the elbows, wrists, forearms, thighs, and ankles. The patient was a carrier of human T cell lymphotropic virus I (HTLV-I), which was probably transmitted from his mother; his mother also had polyarthritis. Histopathologically, the nodules consisted of numerous, small, fibrinoid masses. The synovium adjacent to the tendon sheath was hyperplastic, with fibrinoid necrosis mimicking rheumatoid synovium. However, synovitis was not present inside the adjacent joint. HTLV-I proviral DNA was detected in the cells of the nodule, in tenosynovial cells, and in peripheral blood lymphocytes, but not in skin fibroblasts. In situ reverse transcription assay showed a high quantity of tax/rex messenger RNA in the proliferating lining cells. Based on these features, we classified this case as an atypical manifestation of HTLV-I-associated arthropathy associated with fibrinoid nodules resulting from chronic tenosynovitis.

Neurofibromatosis 2 (NF2) is an autosomal dominant inherited disorder that predisposes to benign tumors of the nervous system as well as a variety of ocular abnormalities. In contrast to NF1, NF2 is associated with only minor developmental abnormalities. The human NF2 gene encodes a tumor suppressor protein, termed schwannomin or merlin, which is a member of a superfamily of proteins thought to link cytoskeletal elements to cell membrane components. To determine the pattern of NF2 gene expression in mouse embryos, we sequenced the mouse NF2 gene and used in situ hybridization and antischwannomin antibodies to determine the developmental expression of the NF2 gene. Schwannomin was detected in most differentiated tissues but was undetectable in undifferentiated tissues. In particular, schwannomin was not detectable in mitotic neuroepithelial cells, the perichondrium, the liver, the neocortex, and the ventricular zone of the developing cerebral cortex. In the heart, expression was observed in all developmental stages beginning on embryonic day 8. In the eye, which shows developmental abnormalities in NF2 patients, expression was detected in the cells of the lens and in the pigment epithelium but weakly detected in retinal neurons. The most striking example of tightly regulated NF2 expression was observed in cells migrating from the ventricular zone to the cortical plate on embryonic days 15 and 16. Only cells in the intermediate zone expressed schwannomin, indicating that schwannomin may play an important role in cellular migration.

Doxorubicin (D) is one of the most active agents in the treatment of breast cancer but can be associated with cardiotoxicity (CT) and febrile neutropenia (FN). Epirubicin, a stereoisomer of doxorubicin, is reported to have similar efficacy but reduced toxicity. A retrospective chart audit was performed to estimate the incidence, average length of hospitalisation and resource consumption for the management of CT and FN in 200 patients breast cancer patients receiving equidoses of doxorubicin or epirubicin. Overall, there were three more episodes of CT in the doxorubicin group than in epirubicin patients (five versus two) at a cost of Canadian dollars C$4268/episode. With regard to FN, there were 11 more episodes in the doxorubicin arm (25 versus 14) at a cost of C$5419/episode. The results of the study support the substitution of equidose epirubicin for doxorubicin in women undergoing treatment for malignancies of the breast. Such a policy may result in reduced toxicity-related management costs.

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF.

There is evidence to suggest that single-agent broad spectrum antibacterials may be cost-effective alternatives to combination antibiotics for the empirical management of febrile neutropenia in cancer patients. The objectives of the present study were 2-fold. The first objective was to compare the clinical effectiveness of ceftazidime monotherapy with that of 2 combination antibiotic regimens in cancer patients with febrile neutropenia. The 2 comparator regimens consisted of tobramycin plus piperacillin, either with (regimen 'CAP') or without (regimen 'AP') cefazolin. The second objective was to perform a cost-effectiveness analysis of the 3 regimens. Meta-analysis of randomised comparative trials between the 3 therapy groups was performed to determine the average overall response rate after 3 to 5 days of treatment. Seven clinical studies were selected for analysis. The overall incidence of adverse drug reactions (ADRs) was determined using the results of comparative and noncomparative studies. A comparative cost-analytic model was applied from a hospital perspective. The costs of primary therapy, hospitalisation, laboratory tests, routine patient care and treating ADRs were calculated, as were future costs. Monotherapy with ceftazidime was associated with an overall response rate of 63.5% and mean per-patient costs of $Can12,000 to $Can14,000. In comparison, regimen AP was associated with an overall response rate of 58.8% and mean costs of $Can13,000 to $Can16,000 per patient. The overall response rate in patients receiving CAP was 75.3%, and the mean cost per patient was $Can11,000 to $Can12,000. Thus, regimen CAP was the most cost-effective therapy from a hospital perspective.

Studies of the trophic activities of brain-derived neurotrophic factor and neurotrophin-3 indicate that both molecules support the survival of a number of different embryonic cell types in culture. We have shown that mRNAs for brain-derived neurotrophic factor and neurotrophin-3 are localized to specific ventral mesencephalic regions containing dopaminergic cell bodies, including the substantia nigra and ventral tegmental area. In the present study, in situ hybridization with 35S-labeled cRNA probes for the neurotrophin mRNAs was combined with neurotoxin lesions or with immunocytochemistry for the catecholamine-synthesizing enzyme tyrosine hydroxylase to determine whether the dopaminergic neurons, themselves, synthesize the neurotrophins in adult rat midbrain. Following unilateral destruction of the midbrain dopamine cells with 6-hydroxydopamine, a substantial, but incomplete, depletion of brain-derived neurotrophic factor and neurotrophin-3 mRNA-containing cells was observed in the ipsilateral substantia nigra pars compacta and ventral tegmental area. In other rats, combined in situ hybridization and tyrosine hydroxylase immunocytochemistry demonstrated that the vast majority of the neurotrophin mRNA-containing neurons in the substantia nigra and ventral tegmental area were tyrosine hydroxylase immunoreactive. Of the total population of tyrosine hydroxylase-positive cells, double-labeled neurons constituted 25-50% in the ventral tegmental area and 10-30% in the substantia nigra pars compacta, with the proportion being greater in medial pars compacta. In addition, tyrosine hydroxylase/neurotrophin mRNA coexistence was observed in neurons in other mesencephalic regions including the retrorubral field, interfascicular nucleus, rostral and central linear nuclei, dorsal raphe nucleus, and supramammillary region. The present results demonstrate brain-derived neurotrophic factor and neurotrophin-3 expression by adult midbrain dopamine neurons and support the suggestion that these neurotrophins influence dopamine neurons via autocrine or paracrine mechanisms. These data raise the additional possibility that inappropriate expression of the neurotrophins by dopaminergic neurons could contribute to the neuropathology of disease states such as Parkinson's disease and schizophrenia.

Isotopic and colorimetric in situ hybridization techniques were combined to determine if nerve growth factor (NGF) mRNA is colocalized with mRNA for the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) in adult rat hippocampus. Quantification of neurons labeled with both 35S-labeled GAD67 mRNA and digoxigenin-labeled NGF cRNA determined that of the NGF cRNA-labeled neurons, 97% within regions CA3-CA1, and 88% within the hilus, were also labeled with GAD67 cRNA. Overall, 47% of the total population of GAD67 cRNA labeled cells were NGF cRNA positive. The greater portion of stratum granulosum was lightly labeled by the NGF cRNA alone. The results indicate that, excepting stratum granulosum, NGF is predominantly synthesized by GABAergic neurons in rat hippocampus.