High Levels Of Immunoreactivity Research Articles

Immunogenicity and immunoreactivity of Tp0821 recombinant protein from Treponema pallidum

Treponemapallidum (Tp) is responsible for invading reproductive organs and the skin in early stages, and involves almost all organs/systems at advanced stages. In the present study, screening of the dominant epitope fragment of the Tp outer membrane protein, Tp0821, was performed and the prokaryotic expression vector pQE32/Tp0821 was constructed. The denaturation and dialysis of rTp0821 were achieved through ultrasound, inclusion body washing and dissolution. Experiments in purified rTp0821‑immune New Zealand rabbits indicated that the recombinant proteins were of high immunogenicity, and the irritation led a marked humoral immune response in the New Zealand rabbits. Western blot analysis showed that the purified recombinant proteins reacted with the Tp‑positive infected serum, confirming the high level of immunoreactivity. The delayed type hypersensitivity of rTp0821 recombinant proteins was positive, indicating that rTp0821 induced a specific cell immune response and was selected as a Tp vaccine candidate protein. The findings of the present study provided novel evidence, which provided information for further investigations on the pathogenic mechanism of Tp and the development of diagnostic reagents.

Estrogen's Effects on Excitatory Synaptic Transmission Entail Integrin and TrkB Transactivation and Depend Upon β1-integrin function.

Estradiol (E2) perfusion rapidly increases the strength of fast excitatory transmission and facilitates long-term potentiation in the hippocampus, two effects likely related to its memory-enhancing properties. Past studies showed that E2's facilitation of transmission involves activation of RhoA signaling leading to actin polymerization in dendritic spines. Here we report that brief exposure of adult male hippocampal slices to 1 nM E2 increases the percentage of postsynaptic densities associated with high levels of immunoreactivity for activated forms of the BDNF receptor TrkB and β1-integrins, two synaptic receptors that engage actin regulatory RhoA signaling. The effects of E2 on baseline synaptic responses were unaffected by pretreatment with the TrkB-Fc scavenger for extracellular BDNF or TrkB antagonism, but were eliminated by neutralizing antisera for β1-integrins. E2 effects on synaptic responses were also absent in conditional β1-integrin knockouts, and with inhibition of matrix metalloproteinases, extracellular enzymes that generate integrin ligands. We propose that E2, acting through estrogen receptor-β, transactivates synaptic TrkB and β1-integrin, and via mechanisms dependent on integrin activation and signaling, reversibly reorganizes the spine cytoskeleton and thereby enhances synaptic responses in adult hippocampus.

Biologically Inactive Leptin and Early-Onset Extreme Obesity

Mutations in the gene encoding leptin (LEP) typically lead to an absence of circulating leptin and to extreme obesity. We describe a 2-year-old boy with early-onset extreme obesity due to a novel homozygous transversion (c.298G→T) in LEP, leading to a change from aspartic acid to tyrosine at amino acid position 100 (p.D100Y) and high immunoreactive levels of leptin. Overexpression studies confirmed that the mutant protein is secreted but neither binds to nor activates the leptin receptor. The mutant protein failed to reduce food intake and body weight in leptin-deficient ob/ob mice. Treatment of the patient with recombinant human leptin (metreleptin) rapidly normalized eating behavior and resulted in weight loss.

Prostaglandin F receptor expression in intrauterine tissues of pregnant rats

In this investigation, we studied the expression and localization of rat prostaglandin F (FP) receptor in uterine tissues of rats on gestational Days 10, 15, 18, 20, 21, 21.5 and postpartal Days 1 and 3 using Western blotting analysis, real-time PCR, and immunohistochemistry. A high level of immunoreactivity was observed on gestational Days 20, 21, and 21.5 with the most significant signals found on Day 20. FP receptor protein was expressed starting on gestational Day 15, and a fluctuating unsteady increase was observed until delivery. Uterine FP receptor mRNA levels were low between Days 10 and 18 of gestation (p < 0.05). The transcript level increased significantly on Day 20 and peaked on Day 21.5 just before labor (p < 0.05). There was a positive correlation between FP receptor mRNA expression and serum estradiol levels (rs = 0.78; p < 0.01) along with serum estradiol/progesterone ratios (rs = 0.79; p < 0.01). In summary, we observed an increase FP receptor expression in rat uterus with advancing gestation, a marked elevation of expression at term, and a concominant decrease during the postpartum period. These findings indicate a role for uterine FP receptors in the mediation of uterine contractility at term.

Spinal cord injury induced changes of nuclear receptors PPARα and LXRβ and modulation with oleic acid/albumin treatment

In previous studies with animal models of spinal cord injury (SCI) pharmacological activation of peroxisome proliferator activated receptors (PPAR) and liver X receptors (LXR) were used to reduce tissue damage and promote behavioral recovery in animal models. We have studied the endogenous expression of the transcription factors PPARα and LXRβ in the chronic stage after SCI in rats. The immunohistochemical investigation revealed a long lasting increase in the level of PPARα in white matter in the vicinity of the lesion site. The source of this signal was identified in a subpopulation of astrocytes outside of the glial scar area. Intrathecal injections of oleic acid/albumin reduced the lesion-induced PPARα immunoreactivity. In addition, ependymal cells displayed a prominent PPARα signal in the non-injured spinal cord, and continued to express the receptor as they proliferated and migrated within the damaged tissue. The nuclear receptor LXRβ was detected at similar levels after SCI as in sham operated animals. We found high levels of immunoreactivity in the gray matter, while in the white matter it was present in subpopulations of astrocytes and oligodendrocytes. Macrophages that had accumulated within the center of the lesion contained LXRβ in their cell nuclei. Possible endogenous functions of PPARα and LXRβ after SCI are discussed, specifically the control of fatty acid and cholesterol metabolism and the regulation of inflammatory reactions.

The effect of enzymatic modification and genetic background on wheat gliadin immunological properties

The immune response to gliadins extracted from 35 wheat cultivars, strains and species was investigated. With the use of a non-competitive ELISA method, we sought to examine if immune reaction occurs between wheat proteins and sera from patients sensitive to gluten, as well as between wheat proteins and rabbit antibodies against pentapeptide QQQPP. Results showed considerable differences among cultivars. Breeding strains STH 183, STH 650, STH 2804, Triticum aethiopicum and Triticum durum showed the lowest immunoreactivity of the native samples. Seventeen of selected wheat flour samples were subjected to enzymatic modifications by subtilisin and transglutaminase. Samples modified by subtilisin exhibited the most extensive change in the level of immunoreactivity of gliadin proteins. The lowest immunoreactivity was reported for the breeding strains STH 1596, STH 2804 and cultivars Tonacja and Zyta. One wheat cultivar (Tonacja), known for its high native gliadin immunoreactivity, was shown to be very susceptible to enzymatic digestion, which resulted in a reduction of its initial immunoreactivity level to low values. Immunoreactivity analysis was also conducted on extracted gliadin fractions from winter wheat strain Ostka strzelecka. The highest level of immunoreactivity to both types of antibodies we used was indicated for ω5-gliadins. It was, however, abrogated in the aftermath of enzymatic modifications.

Differential Expressions of Synaptogenic Markers between Primary Cultured Cortical and Hippocampal Neurons

Primary dissociated neuronal cultures are widely used research tools to investigate of pathological mechanisms and to treat various central and peripheral nervous system problems including trauma and degenerative neuronal diseases. We introduced a protocol that utilizes hippocampal and cortical neurons from embryonic day 17 or 18 mice. We applied appropriate markers (GAP-43 and synaptophysin) to investigate whether neurite outgrowth and synaptogenesis can be distinguished at a particular period of time. GAP-43 was found along the neural processes in a typical granular pattern, and its expression increased proportionally as neurites lengthened during the early in vitro period. Unlike GAP-43, granular immunoreactive patterns of synaptophysin along the neurites were clearly found from day 2 in vitro with relatively high immunoreactive levels. Expression of synaptic markers from cortical neurons reached peak level earlier than that of hippocampal neurons, although neurite outgrowths of hippocampal neurons were faster than those of cortical neurons. The amount of peak synaptic markers expressed was also higher in cortical neurons than that in hippocampal neurons. These results strongly suggest the usefulness of primary cultured neurons from mice embryos for synaptic function and plasticity studies, because of their clear and typical patterns of morphology that establish synapses. Results from this study also suggest the proper amount of time in vitro according to neuronal types (cortical or hippocampal) when utilized in experiments related with synaptogenesis or synaptic activities.

Research Review

In previous studies with animal models of spinal cord injury (SCI) pharmacological activation of peroxisome proliferator activated receptors (PPAR) and liver X receptors (LXR) were used to reduce tissue damage and promote behavioral recovery in animal models. We have studied the endogenous expression of the transcription factors PPARα and LXRβ in the chronic stage after SCI in rats. The immunohistochemical investigation revealed a long lasting increase in the level of PPARα in white matter in the vicinity of the lesion site. The source of this signal was identified in a subpopulation of astrocytes outside of the glial scar area. Intrathecal injections of oleic acid/albumin reduced the lesion-induced PPARα immunoreactivity. In addition, ependymal cells displayed a prominent PPARα signal in the non-injured spinal cord, and continued to express the receptor as they proliferated and migrated within the damaged tissue. The nuclear receptor LXRβ was detected at similar levels after SCI as in sham operated animals. We found high levels of immunoreactivity in the gray matter, while in the white matter it was present in subpopulations of astrocytes and oligodendrocytes. Macrophages that had accumulated within the center of the lesion contained LXRβ in their cell nuclei. Possible endogenous functions of PPARα and LXRβ after SCI are discussed, specifically the control of fatty acid and cholesterol metabolism and the regulation of inflammatory reactions.

Rapid, Simple, Quantitative, and Highly Sensitive Antibody Detection for Lyme Disease

There is currently a need for improved serological tests for the diagnosis and monitoring of Lyme disease, an infection caused by Borrelia burgdorferi. In the present study, we evaluated luciferase immunoprecipitation systems (LIPSs) for use for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. Initially, serum samples from a cohort of patients and controls (n = 46) were used for training and were profiled by the use of 15 different B. burgdorferi antigen constructs. For the patient sera, the antibody responses to several B. burgdorferi antigens, including VlsE, flagellin (FlaB), BmpA, DbpA, and DbpB, indicated that the antigens had high levels of immunoreactivity. However, the best diagnostic performance was achieved with a synthetic protein, designated VOVO, consisting of a repeated antigenic peptide sequence, VlsE-OspC-VlsE-OspC, Analysis of an independent set of serum samples (n = 139) used for validation showed that the VOVO LIPS test had 98% sensitivity (95% confidence interval [CI], 93% to 100%; P < 0.0001) and 100% specificity (95% CI, 94% to 100%; P < 0.0001). Similarly, the C6 peptide enzyme-linked immunosorbent assay (ELISA) also had 98% sensitivity (95% CI, 93% to 100%; P < 0.0001) and 98% specificity (95% CI, 90% to 100%; P < 0.0001). Receiver operating characteristic analysis revealed that the rates of detection of Lyme disease by the LIPS test and the C6 ELISA were not statistically different. However, the VOVO LIPS test displayed a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution. These results suggest that screening by the LIPS test with VOVO and other B. burgdorferi antigens offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.

Abstract A178: Role of tumor and host-derived HGF in drug resistance to EGFR inhibitors in EGFR activating mutation-positive lung cancer

Abstract Background: Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations (EGFRmu) responds favorably to the EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or erlotinib. However, 25–30% of patients with EGFRmu show intrinsic resistance, and the responders invariably acquire resistance to EGFR-TKIs. Here, we examined the role of hepatocyte growth factor (HGF), a specific ligand of MET, in gefitinib resistance of NSCLC with EGFRmu. Methods: NSCLC cells, PC-9 and HCC827, with EGFRmu, but not T790M second mutation in EGFR or MET amplification, were used in this study. Sensitivity to gefitinib was determined by MTT assay. Phosphorylation of MET, EGFR, ERBB3, and the PI3K/Akt pathway were examined by Western blotting. HGF expression in 20 tumors from 16 NSCLC patients with EGFRmu who were treated with gefitinib was determined by immunohistochemistry. Results: HGF induced resistance to gefitinib by restoring Akt phosphorylation independently of ErbB3. Specific down-regulation of MET, but not ErbB3, reversed gefitinib resistance and Akt phosphorylation induced by HGF. High levels of immunoreactivity for HGF were detected in cancer cells of 3 of 3 intrinsic resistant tumors and in one of 2 tumors with acquired resistance without T790M second mutation or MET amplification, suggesting HGF-induced resistance by autocrine mechanism. The lung cancer cells also became resistant to EGFR-TKIs when co-cultured in vitro with HGF-producing fibroblasts and co-injected into SCID mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4 successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo. Co-localization of fibroblasts and HGF was detected in both xenograft tumors in mouse model and lung cancer patient specimens, suggesting HGF-induced resistance by paracrine mechanism. Conclusions: The findings indicate that HGF-mediated MET activation is a novel mechanism of intrinsic and acquired gefitinib-resistance in NSCLC with EGFRmu by both autocrine and paracrine mechanisms. Therefore, inhibition of HGF-MET signaling may be considerable strategy for more successful treatment with EGFR-TKIs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A178.

Differential localization of γ‐aminobutyric acid type a and glycine receptor subunits and gephyrin in the human pons, medulla oblongata and uppermost cervical segment of the spinal cord: An immunohistochemical study

Gephyrin is a multifunctional protein responsible for the clustering of glycine receptors (GlyR) and gamma-aminobutyric acid type A receptors (GABA(A)R). GlyR and GABA(A)R are heteropentameric chloride ion channels that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of gephyrin and the major GABA(A)R and GlyR subunits in the human light microscopically in the rostral and caudal one-thirds of the pons, in the middle and caudal one-thirds of the medulla oblongata, and in the first cervical segment of the spinal cord. The results demonstrate a widespread pattern of immunoreactivity for GlyR and GABA(A)R subunits throughout these regions, including the spinal trigeminal nucleus, abducens nucleus, facial nucleus, pontine reticular formation, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, lateral cuneate nucleus, and nucleus of the solitary tract. The GABA(A)R alpha(1) and GlyR alpha(1) and beta subunits show high levels of immunoreactivity in these nuclei. The GABA(A)R subunits alpha(2), alpha(3), beta(2,3), and gamma(2) present weaker levels of immunoreactivity. Exceptions are intense levels of GABA(A)R alpha(2) subunit immunoreactivity in the inferior olivary complex and high levels of GABA(A)R alpha(3) subunit immunoreactivity in the locus coeruleus and raphe nuclei. Gephyrin immunoreactivity is highest in the first segment of the cervical spinal cord and hypoglossal nucleus. Our results suggest that a variety of different inhibitory receptor subtypes is responsible for inhibitory functions in the human brainstem and cervical spinal cord and that gephyrin functions as a clustering molecule for major subtypes of these inhibitory neurotransmitter receptors.

Structural Differences in the Umbilical Vein Wall after Full-Term and Pre-term Delivery

With the exception of its most proximal segment, the human umbilical cord lacks innervation. It might be expected, therefore, that a paracrine effect through the direct contact between the smooth muscle cells and the endothelium may be particularly important in the control of the fetoplacental circulation. In this study, electron microscopy and immunohistochemistry were applied to examine umbilical veins immediately after full-term and pre-term delivery. The smooth muscle cells in the upper layer of the tunica media exhibited long, foot-like processes with c-kit immunoreactivity. In the umbilical vein of full-term neonates more than 50% of these cell processes display a normal ultrastructure and they were closely associated with the lamina elastica interna. Whereas in pre-term infants more than 60% of these cell processes exhibit signs of severe shrinkage and detachedness from the lamina elastica interna. At the same time, the high level of immunoreactivity of the endothelial cells as regards the proapoptotic gene product Bax in pre-term infants is indicative of an enhanced apoptotic process in these cells.

The expression of the Goodpasture antigen-binding protein (ceramide transporter) in adult rat brain

The Goodpasture antigen-binding protein (GPBP) plays a critical role in brain development. Knockdown of GPBP leads to loss of myelinated tracts in the central nervous system and to extensive apoptosis in the brain during early embryogenesis. GPBP was initially identified as a protein associated with the autoantigen in Goodpasture autoimmune syndrome, where it was shown to be a kinase that regulates type IV collagen organization. GPBP isoforms bind and transport ceramide from the endoplasmic reticulum to the Golgi apparatus and are therefore also known as ceramide transporters (CERT). Ceramide dysregulation is involved in autoimmunity and neurodegenerative disorders. In order to analyze the possible role of GPBP in neuroinflammation and neurodegeneration we studied the basal GPBP expression in normal rat brain. High levels of immunoreactivity were detected in neurons of the cerebral cortex, hippocampal formation, the basal ganglia, the olfactory bulb and nuclei of the thalamus, the hypothalamus and the septal area. Lower expression levels of GPBP were observed widely throughout the brain, suggesting that GPBP plays an important role in central nervous system neuron function.

Hepatocyte growth factor as inducer of gefitinib resistance in non-small cell lung cancer harboring EGFR activating mutations

e19034 Background: Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations (EGFRmu) responds favorably to the EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or erlotinib. However, 25–30% of patients with EGFRmu show intrinsic resistance, and the responders invariably acquire resistance to EGFR-TKIs. Here, we examined the role of hepatocyte growth factor (HGF), a specific ligand of MET, in gefitinib resistance of NSCLC with EGFRmu. Methods: NSCLC cells, PC-9 and HCC827, with EGFRmu, but not T790M second mutation in EGFR or MET amplification, were used in this study. Sensitivity to gefitinib was determined by MTT assay. Phosphorylation of MET, EGFR, ERBB3, and the PI3K/Akt pathway were examined by Western blotting. HGF expression in 20 tumors from 16 NSCLC patients with EGFRmu who were treated with gefitinib was determined by immunohistochemistry. Results: HGF induced resistance to gefitinib by restoring Akt phosphorylation independently of ErbB3. Specific down-regulation of MET, but not ErbB3, reversed gefitinib resistance and Akt phosphorylation induced by HGF. Importantly, high levels of immunoreactivity for HGF were detected in cancer cells of 3 of 3 intrinsic resistant tumors and in one of 2 tumors with acquired resistance without T790M second mutation or MET amplification. Conclusions: The findings indicate that HGF-mediated MET activation is a novel mechanism of intrinsic and acquired gefitinib-resistance in NSCLC with EGFRmu. Therefore, inhibition of HGF-MET signaling may be considerable strategy for more successful treatment with EGFR-TKIs. [Table: see text]

Changes in mouse mu opioid receptor Exon 7/8‐like immunoreactivity following food restriction and food deprivation in rats

Opioid agonists and antagonists respectively increase and decrease food intake. That selective mu opioid antagonists are more effective than antisense probes directed against the mu opioid receptor (MOR-1) gene in reducing deprivation-induced feeding suggests a role for isoforms. Both food restriction and deprivation alter protein and mRNA levels of opioid peptides and receptors. Antisera directed against Exon 4 of the MOR-1-like immunoreactivity (LI) (Exon 4) clone or directed against mouse Exons 7/8 (mE7/8-LI) revealed high levels of immunoreactivity in brain nuclei related to feeding behavior. Therefore, the present study assessed MOR-1LI and mE7/8-LI in hypothalamic and extrahypothalamic sites in rats exposed to ad libitum feeding, food restriction (2, 7, 14 days), or food deprivation (24, 48 h). MOR-1-LI displayed robust reactivity, but was insensitive to food restriction or deprivation. mE7/8-LI, both in terms of cell counts and relative optical density, was significantly and selectively increased in the dorsal and ventral parvocellular subdivisions of the hypothalamic paraventricular nucleus in food-restricted (14 days) rats, but all other restriction or deprivation regimens were ineffective in other hypothalamic nuclei. In contrast, significant and site-specific decreases in relative optical density in the rostral part of the nucleus tractus solitarius (NTS) were observed in food-restricted (2, 7 days) or food-deprived (24, 48 h) animals, but these regimens were ineffective in the other extrahypothalamic sites. This study indicates the sensitivity of this mE7/8-LI probe in the hypothalamic parvocellular paraventricular nucleus and rostral NTS to food restriction and deprivation in rats.

Development of sympathetic glia in mice

Two sorts of glial cell are associated with sympathetic ganglia — Schwann cells, which are associated with peripheral nerves, and sympathetic (satellite) glia, which support neurons in ganglia. The development of sympathetic neurons has been very well studied over many years, but surprisingly little is known about the development of sympathetic glia. We examined the development of sympathetic glia in mouse sympathetic ganglia in both wild type and transgenic mice. Previous studies have shown that brain-derived fatty acid binding protein (B-FABP) is a marker of glial precursors. In wild type mice, B-FABP+cells were first detected immunohistochemically at E11.5. The first BFABP+cells appeared in the most superficial layer of the ganglion, but by E12.5 they were also present deeper in the ganglion, where they continued to increase in number until the adult density and appearance was reached by E16.5. B-FABP was never colocalised with immunoreactivity to tyrosine hydroxylase at any embryonic stage. A marker of mature peripheral glial cells, S100b, did not appear until E15.5. In heterozygote mice in which green fluorescent protein (GFP) expression is driven by the Ret-promoter, B-FABP was never co-localised with GFP. However, sympathetic glial precursors did express high levels of immunoreactivity for GDNF family ligand receptor alpha 1 (GFRalpha1) between E10.5 and E14.5. GDNF-GFRalpha1 can utilise neural cell adhesion molecule (NCAM) rather than Ret as the transmembrane signalling component. NCAM was present in B-FABP immunoreactive cells. An examination of GDNF null mutants suggested that there were no obvious consequences to satellite glial development of the loss of GDNF.

Up‐regulation of a BCL9‐related β‐catenin‐binding protein, B9L, in different stages of sporadic colorectal adenoma

Aberrant activation of Wnt signaling is a critical event in the development of human colorectal tumors. The aim of this study was to elucidate the role of B9L and its association with beta-catenin in each stage of the adenoma-carcinoma sequence of human colorectal tumorigenesis. We investigated the expression levels of B9L in sporadic colorectal adenomas and carcinomas, categorized according to the Vienna classification, using real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemical analysis. B9L was expressed in the nuclei of non-neoplastic colonic mucosa cells and was overexpressed in the nuclei of neoplasias and invasive carcinoma cells. Immunoreactivity to B9L protein was correlated with the progressive grades of colorectal neoplasias, as was the expression of B9L mRNA. A high level of immunoreactivity to nuclear B9L was present in 27% of low-grade neoplasias and in more than 50% of high-grade neoplasias and invasive carcinomas, whereas a high level of nuclear B9L immunoreactivity was not observed in any of the non-neoplastic mucosa samples. The expression of B9L was dramatically increased in low-grade neoplasias compared with that in non-neoplastic mucosa. B9L may play an important role in tumorigenesis induced by aberrant activation of Wnt signaling and may act as a key protein in the progressive dysplasia of adenoma.

Urinary Progestogen and Estrogen Excretion During Pregnancy in Eulemur macaco flavifrons, E. rubriventer, and Hapalemur griseus occidentalis

Via a non-invasive approach, we aimed to provide comparative data on the presence and relative abundance of progestogen and estrogen metabolites excreted into the urine of Eulemur rubriventer, E. macaco flavifrons and Hapalemur griseus occidentalis and to characterize the patterns of progestogen and estrogen excretion during pregnancy. We found that estrone is the major urinary estrogen in Eulemur macaco flavifrons and Hapalemur griseus occidentalis, while 16α-hydroxyestrone appeared to be the predominant estrogen in the urine of E. rubriventer. Estradiol-17s was either absent (Hapalemur) or present in very low amounts. HPLC of progestogens had high levels of immunoreactivity in an assay against 5α-pregnane-3α-ol-20-one (5-P-3OH) in each species, though the nature of the progestogens measured by the 5-P-3OH assay differed among species. During pregnancy, 5-P-3OH levels remained relatively low in Eulemur rubriventer, whereas in E. macaco flavifrons and Hapalemur griseus occidentalis, a sustained elevation in levels occurred from mid-pregnancy until the final weeks before parturition. Estrogen excretion differed depending on the sex of the fetus in each species, with only females carrying male infants showing clearly elevated estrogen levels during the last 6–8 weeks of gestation. In conclusion, we demonstrate both species similarities and differences in the metabolism and urinary excretion patterns of reproductive hormones throughout gestation in the Lemuridae. The data not only extend our knowledge on the reproductive physiology of lemurs but also show that more studies on other lemur taxa are needed to provide a broader basis for interspecific comparison.

Fluorophore-Assisted Retinal Break Detection Using Antibodies to Glial Fibrillary Acidic Protein

To evaluate the role of fluorescent antibodies as a means of enhancing the detection of retinal breaks during vitrectomy for rhegmatogenous retinal detachment. In ex vivo studies, unfixed, porcine retinal flatmounts were incubated with Cy3 anti-GFAP. Experiments were repeated in the presence of excess soluble GFAP and after surface excimer laser ablation through the internal limiting membrane, into the Müller cell foot processes. Tissue was also incubated with trypan blue, and cross-species immunoreactivity was determined in bovine, rabbit, and human retina. In vivo studies were conducted in a porcine model of rhegmatogenous retinal detachment. Cy3 anti-GFAP was injected into the vitreous cavity of eyes with retinal breaks and then rinsed from the eye. Barrier filters were fitted to the operating microscope to allow intraoperative visualization of tissue stained with Cy3. Excitation endoillumination was provided by a 532-nm diode-pumped laser. In ex vivo studies, retinal flatmounts exposed to Cy3 anti-GFAP showed minimal surface fluorescence, but exposed glial elements at the cut edge of the flatmount stained brightly, as did those exposed by excimer ablation of the Müller cell membrane. Blocking studies confirmed that binding was antigen specific. Trypan blue colocalized to the cut edge of retinal flatmounts. All species showed high levels of immunoreactivity except rabbit. In vivo studies demonstrated selective intraoperative staining of retinal breaks with a high level of specificity. Intraoperative vital staining of retinal breaks is possible in an animal model of retinal detachment. Ex vivo studies indicate that this occurs because the Cy3 anti-GFAP selectively binds the intermediate filaments of glial cells with damaged or destroyed cell membranes.

Transforming growth factor-beta- and Activin-Smad signaling pathways are activated at distinct maturation stages of the thymopoeisis.

Members of the transforming growth factor (TGF)-beta family play pivotal roles in the control of differentiation, proliferation and tolerance in peripheral T cells. Recently, they have been implicated in thymic selection, but their role is so far not well characterized. In the present study, we demonstrate that specific thymocyte populations are under the influence of either the TGF-beta and/or Activin pathway, and transduce signals into the nucleus via phosphorylated Smad2 (pSmad2). Thymocytes in the medulla and in the subcapsular zone expressed nuclear translocated pSmad2, a hallmark of active TGF-beta/Activin receptor signaling. When analyzed at the cellular level, the pSmad2(+) cells were confined to the double-negative (DN) and single-positive (SP) subpopulations. Moreover, the most immature DN thymocytes (CD44(+)CD25(-) and CD44(+)CD25(+)) expressed higher levels of pSmad2 compared to the more mature DN. In vitro stimulation demonstrated that pure CD44(+)CD25(-), CD44(+)CD25(+) and CD44(+)CD25(+) thymocytes respond to ActivinA, while the mature CD4(+) and CD8(+) SP thymocytes respond to TGF-beta stimulation measured as enhanced phosphorylation of Smad2. Double staining of pSmad2(+) cells with either the Activin type I receptor, ALK4, or the TGF-beta type I receptor, ALK5, demonstrated that pSmad2(+) DN cells exhibited high levels of immunoreactivity to ALK4 and moderate levels of immunoreactivity to the TGF-beta-responsive ALK5 receptor. In sharp contrast, the SP pSmad2(+) cells were predominately ALK5(+). Collectively, our results demonstrate that early and late thymocytes express pSmad2 in the nuclei in vivo. The functional experiments in vitro suggest that members of the TGF-beta family (TGF-beta or Activin) may play important non-redundant roles during different stages of thymopoiesis.