The dependency of bioactive follicle-stimulating hormone secretion on gonadotropin-releasing hormone in hypogonadal and cycling women

An antagonist analog of GnRH, (Ac-delta 3-Pro1,p-F-D-Phe2,D-Trp3,6)GnRH (4F-antagonist), was administered to normal women and women with hypergonadotropic hypogonadism. Serum FSH levels were determined by both the granulosa cell aromatase bioassay and RIA. The constant infusion of 4F-antagonist (30 micrograms/kg.h) to the four hypogonadal women resulted in a more pronounced decline in bioactive FSH (62%) than in immunoreactive FSH levels (30%), and the FSH bioactive to immunoreactive ratio decreased significantly (P less than 0.05). Infusion of 4F-antagonist in normal women in the midfollicular phase revealed a similar pattern of suppression of bioactive (64%) and immunoreactive FSH (29%). When 4F-antagonist was administered sc at a dose of 80 micrograms/kg twice daily for 3 days to normal women in the midfollicular phase of their cycles, the bioactive FSH response was biphasic, with the maximal decrease on the second day, followed by return to basal levels on the third day. Correspondingly, there was a precipitous decline in serum estradiol (apparent demise of the dominant follicle), followed by a progressive rise in estradiol levels. Thus, in contrast to immunoreactive FSH levels, bioactive FSH more clearly reflects the biological action of FSH on the follicle in response to GnRH antagonist administration in women. The disparity in the quantitative decline between serum bioactive and immunoreactive FSH levels after presumed blockade of the GnRH receptor may reflect the microheterogeneity of the FSH molecule and suggests that alterations in the biological activity of secreted FSH may be GnRH dependent.     

160例成人HIV感染者/AIDS患者机会性感染与CD4+之间关系分析

目的：分析中国成人艾滋病病毒（HIV）感染者／艾滋病（AIDS）患者机会性感染发生的频率与CD4^ 细胞数之间的关系。方法：对1990－2001年在北京佑安医院就诊的160例成人HIV感染者／AIDS患者CD4^ 、CD8^ 进行跟踪分析。结果：（1）CD4^ ＞500个／μl66人次（12．7％），CD4^ 为200个／μl-500个／μl212人次（41．1％），CD4^ ＜200个／μl234人次（45．3％）。在CD4^ ＜个200／μl中，CD4^ ＜100个／μl128人次（24．8％），CD4^ ＜50个／μl89人次（17．2％）。（2）CD4^ ＞200个／μl时，共发生机会性感染33人次（15．6％）。CD4^ ＜200个／μl时，共发生机会性感染170人次（72．6％），CD4^ 为100个／μl0－200个／μl之间发生机会性感染42人次（39．6％），CD4^ ＜100个／μl发生机会性感染128人次（98．4％），其中CD4^ ＜50个／μl发生机会性感染87人次（97．8％）。结论：中国成人HIV感染者／AIDS患者在CD4^ ＞200个/μl时机会性感染出现频率较少，CD4^ ＜200个／μl时机会性感染的频率明显增加，CD4^ ＜100个／μl和＜50个／μl时，感染率约为100％。     

RANKL和OPG在不同骨移植材料拔牙位点保存区的表达

目的 通过观察破骨细胞核因子kB受体活化因子配体(receptor activator nuclear factor kappa B ligand,RANKL)和骨保护因子(osteoprotegerin,OPG)在不同骨移植材料拔牙位点保存时骨构建过程中表达变化,探讨破骨细胞对不同骨移植材料植入拔牙窝内在引导骨再生中的作用.方法 选取6只12月龄雄性beagle犬,微创拔除上下颌双侧第一侧切牙,将4个位点随机做不同处理,A组填人富血小板纤维蛋白(platelet-rich fibrin,PRF),B组填入骨诱导活性材料(osteoinduction active material,OAM),C组填入羟基磷灰石生物陶瓷(coralline hydroxyapatite,CHA),D组空置(对照).6只犬随机分别于术后4周和12周各处死3只,制作牙槽骨标本.免疫组化检测24个拔牙位点保存制作的犬牙槽骨标本中RANKL与OPG的表达变化.结果 术后4周骨组织中OPG的表达A组高于B组、C组和D组(P＜0.05),RANKL的表达D组高于C组、B组和A组(P＜0.05).术后12周骨组织中OPG的表达D组高于C组、B组和A组(P＜0.05),RANKL的表达C组高于B组、A组和D组(P＜0.05).结论 骨移植材料在骨改建过程中通过促进成骨细胞分化成熟而刺激OPG表达升高,抑制破骨细胞.提示骨移植材料通过调控RANKL和OPG在拔牙窝新骨形成过程中发挥作用.     

Heterodimeric fly glycoprotein hormone-alpha2 (GPA2) and glycoprotein hormone-beta5 (GPB5) activate fly leucine-rich repeat-containing G protein-coupled receptor-1 (DLGR1) and stimulation of human thyrotropin receptors by chimeric fly GPA2 and human GPB5

Glycoprotein hormones play important roles in thyroid and gonadal function in vertebrates. The glycoprotein hormone alpha-subunit forms heterodimers with different beta-subunits to activate TSH or gonadotropin (LH and FSH) receptors. Recent genomic analyses allowed the identification of another alpha-subunit, GPA2, and another beta-subunit, GPB5, in human, capable of forming heterodimers to activate TSH receptors. Based on comparative genomic searches, we isolated the fly orthologs for human GPA2 and GPB5, each consisting of 10 cysteine residues likely involved in cystine-knot formation. RT-PCR analyses in Drosophila melanogaster demonstrated the expression of GPA2 and GPB5 at different developmental stages. Immunoblot analyses further showed that fly GPA2 and GPB5 subunit proteins are of approximately 16 kDa, and coexpression of these subunits yielded heterodimers. Purified recombinant fly GPA2/GPB5 heterodimers were found to be glycoproteins with N-linked glycosylated alpha-subunits and nonglycosylated beta-subunits, capable of stimulating cAMP production mediated by fly orphan receptor DLGR1 but not DLGR2. Although the fly GPA2/GPB5 heterodimers did not activate human TSH or gonadotropin receptors, chimeric fly GPA2/human GPB5 heterodimers stimulated human TSH receptors. These findings indicated that fly GPA2/GPB5 is a ligand for DLGR1, thus showing the ancient origin of this glycoprotein hormone-seven transmembrane receptor-G protein signaling system. The fly GPA2 also could form heterodimers with human GPB5 to activate human TSH receptors, indicating the evolutionary conservation of these genes and suggesting that the GPA2 subunit may serve as a scaffold for the beta-subunit to activate downstream G protein-mediated signaling.     

BMSCs复合PRF修复牙槽骨缺损中OPG和RANKL的表达及意义

目的：探讨骨髓间充质干细胞（BMSCs）复合富血小板纤维蛋白（PRF）修复牙槽骨缺损中骨保护素（OPG）和核因子B受体活化因子配体（RANKL）的表达及意义。方法：取健康雄性2月龄新西兰兔36只,随机分为A、B、C、D 4组,均在全麻下微创拔除下颌左侧中切牙。 A组植入BMSCs与PRF复合物,B组植入PRF,C组植入BMSCs,D组为空白对照组。按术后4,8,12周3个时间点（每个时间点9只）处死动物并立即于骨缺损部位取材,免疫组织化学方法检测OPG和RANKL的表达。结果：4,8,12周时A组、B组、C组OPG的表达高于D组（P＜0．05）;4,8,12周时A组、B组、C组RANKL的表达高于D组（P＜0．05）;析因分析显示自体BMSCs复合PRF修复牙槽骨缺损中OPG,RANKL的表达高于单独使用BMSCs或PRF（P＜0．05）。结论：自体BMSCs复合PRF修复牙槽骨缺损会增加OPG,RANKL的表达,有助于牙槽骨改建。     

Direct observation therapy with appropriate treatment regimens was associated with a decline in second-line drug-resistant tuberculosis in Taiwan

The resistance of Mycobacterium tuberculosis (MTB) to second-line drugs (SLDs) is growing worldwide; however, associations between the appropriateness of treatment for tuberculosis (TB) and whether the directly observed treatment, short course (DOTS)/DOTS-plus programs had an impact on the prevalence of SLD-resistant MTB are still uncertain. We performed a retrospective analysis of resistance profiles among MTB isolates obtained from 6,035 consecutive patients from 2004 to 2011 at two TB referral hospitals in Taiwan. There was a significant decrease (all p-values <0.01) in the prevalence of MTB isolates that were resistant to fluoroquinolones, injectable SLDs, and orally administered SLDs, and multidrug-resistant (MDR) and extensively drug-resistant (XDR) MTB isolates over time. There was a significant increase in the coverage rate of DOTS/DOTS-plus programs and that of administering appropriate first-line and second-line regimens (all p?<?0.01). Compared with isoniazid-susceptible isolates, high-level (1.0 mg/L) isoniazid-resistant and MDR isolates showed extensive cross resistance to ofloxacin (5.9 %, p?<?0.01 and 33.6 %, p?<?0.01), levofloxacin (9.6 %, p?<?0.01 and 38.1 %, p?<?0.01), moxifloxacin (11.1 %, p?<?0.01 and 26.5 %, p?<?0.01), kanamycin (6.8 %, p?<?0.01 and 16.7 %, p?<?0.01), ethionamide (6.4 %, p?<?0.01 and 16.2 %, p?<?0.01), and para-aminosalicylic acid (13.1 %, p?<?0.01 and 20.4 %, p?<?0.01), but not to capreomycin (2.0 %, p?=?0.06 and 1.6 %, p?=?0.08). The decline in prevalence of resistance to SLDs was negatively correlated with the rise in rates of administering appropriate regimens as well as the DOTS/DOTS-plus programs, but not with the increase in usage of second-line regimens. The implementation of DOTS/DOTS-plus programs with appropriate regimens was associated with a decrease in the prevalence of SLD-resistant and XDR TB.     

Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury

Neuronal calcium (Ca²⁺)-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca²⁺-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca²⁺-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca²⁺-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.Calcium (Ca²⁺) plays a crucial role in many and diverse cellular processes, including neurotransmission (1). Glutamate and neuropeptides are neurotransmitters released from the central terminals of dorsal root ganglion (DRG) neurons in the spinal dorsal horn, where signals for different sensory modalities, including pain, are conveyed to higher centers (2–12). Neurotransmitter release is tightly regulated by Ca²⁺-dependent SNARE proteins whose activity is regulated by Ca²⁺-binding proteins (CaBPs) (1, 7, 13).Parvalbumin (PV), calbindin D-28K (CB), calretinin (CR), and secretagogin (Scgn) are extensively studied EF-hand CaBPs, and they have also emerged as valuable anatomical markers for morphologically and functionally distinct neuronal subpopulations (14–17). The expression of CaBPs in DRG neurons has been thoroughly studied (18). Moreover, neuronal Ca²⁺ sensor 1 and downstream regulatory element-antagonist modulator (DREAM) are also EF-hand Ca²⁺-binding proteins in DRGs and the spinal cord (19, 20). Despite these advances, a CaBP has so far not been characterized in the majority of small- and medium-sized DRG neurons, many of which represent nociceptors.The subfamily of neuronal Ca²⁺-binding proteins (NECABs) consists of three members (NECAB1–NECAB3), probably as a result of gene duplication (21). NECABs are also EF-hand proteins, with one pair of EF-hand motifs in the N terminus and a putative antibiotic biosynthesis monooxygenase domain in the C terminus, which are linked by a NECAB homogeneous region (22). NECAB1/2 are restricted to the nervous system, whereas NECAB3 is also expressed in the heart and skeletal muscle (21).NECAB1 was first identified as the target protein of synaptotagmin I C2A-domain by affinity chromatography, with its expression restricted to layer 4 cortical pyramidal neurons, inhibitory interneurons, and hippocampal CA2 pyramidal cells in mouse brain (21, 23). The gene of the second member was cloned from mouse and initially named Necab. It encodes a 389-aa (NECAB2) (24). NECAB2 was identified as a downstream target of Pax6 in mouse retina, which is involved in retinal development (24, 25), as well as being a binding partner for the adenosine A_2A receptor (22). Furthermore, an interaction between NECAB2 and metabotropic glutamate receptor 5 (mGluR5) was demonstrated in rat hippocampal pyramidal cells, possibly regulating mGluR5’s coupling to its signaling machinery (26). Finally, NECAB3, also known as XB51, was isolated as an interacting target for the neuron-specific X11-like protein and is possibly involved in the pathogenesis of Alzheimer’s disease (27, 28).Very recently, NECAB1/2 were shown to have complementary expression patterns in mouse hippocampus at the mRNA and protein levels, whereas NECAB3 is broadly distributed in the hippocampus (29). NECAB1-expressing cells were seen throughout the cell-sparse layers of Ammon’s horn and the hilus of the dentate gyrus. In contrast, NECAB2 is enriched in pyramidal cells of the CA2 region. A minority of NECAB1⁺ neurons were GABAergic yet did not coexpress PV, CB, or CR (29).Here, we investigated the expression of NECAB1/2 in mouse DRGs and spinal cord using quantitative PCR (qPCR), immunohistochemistry (also combined with CLARITY) (30), and Western blotting. We compared the distribution of NECABs with that of the four CaBPs restricted to neurons, PV, CB, CR, or Scgn. NECAB⁺ neurons in the spinal dorsal horn were phenotyped using transgenic mice harboring genetic markers for excitatory [vesicular glutamate transporter 2 (VGLUT2)] (31) or inhibitory [glutamate decarboxylase 67 (GAD67)] (32) cell identities. Finally, the effect of peripheral nerve injury was analyzed.     

Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC₅₀ of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.Multiple methods have been developed to isolate HIV broadly neutralizing antibodies (bnAbs) (1–12). Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including b12, 2F5, 2G12, 4E10, and Z13 (13–20). These antibodies exhibit a range of neutralization breadth against primary isolates from 30 to 90% but have moderate neutralization potency (median IC₅₀ of ∼2–4 µg/mL). Access to infected donors who have high serum titers of bnAbs (21, 22) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (1–4, 6–8).One of the newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high-throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of the new generation of bnAbs, PG9 and PG16 (1), and since then has revealed several sites of vulnerability to bnAb recognition on HIV envelope (Env) (1–4, 6, 7). An alternative method of bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG⁺ memory B cells of interest by cell sorting (6, 8, 9, 23, 24). However, soluble baits have not been successful in isolating antibody responses targeting quaternary epitopes, including the trimer-apex site surrounding the N160 glycan, because the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell-surface Env, called “GFP-293T^BaL cells,” were used recently to isolate antibodies 3BC176 and 3BC315 (10, 25). These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating the utility of the approach, but the method was reported to be less efficient than the use of soluble protein baits (10, 25).The favorable antigenic profile of the soluble BG505 SOSIP.664 gp140 trimer opens the possibility of its use for isolating quaternary-specific antibodies by single-cell sorting (26). To this end, we used BG505 SOSIP.664 gp140 to select for memory B cells from a donor from whom we previously had isolated the trimer-specific bnAbs PGT141–145 (3, 21). (For naming of PGT and PGDM bnAbs, please see SI Materials and Methods, Antibody Nomenclature.) We describe the isolation of previously unidentified somatic variants that are highly divergent from PGT145 and display a range of neutralization breadth and potency, with some being broader and more potent than the previously described PGT145 family members. Overall, the results reveal a mosaic of antibody responses against the trimer-apex site of vulnerability that have important implications for immunogen design in general and for the future optimization of BG505 SOSIP.664 and related native-like trimers as vaccine candidates.