Pearls for Starting a Headache Surgery Practice in Academic and Private Practice.

There has been a growing body of evidence indicative of the effectiveness of headache surgery in treating patients with refractory headache disorders. The American Society of Plastic Surgeons issued a Policy Statement in 2018 stating that peripheral nerve decompression surgery for the treatment of refractory chronic headache disorders in select patients is considered a standard of care treatment. This endorsement sparked the interest of numerous plastic surgeons into initiating their own headache surgery practices. However, establishing a headache surgery clinic introduces challenges and considerations. This report outlines the key pillars for launching a successful headache surgery practice in academic and private practice environments.

Cumplimiento del tratamiento farmacológico en mujeres adultas con hipotiroidismo primario / Drug Treatment Adherence in Adult Women with Primary Hypothyroidism

Introducción: La falta de cumplimiento al tratamiento puede ser causa del fracaso terapéutico en pacientes hipotiroideos. Objetivos: Conocer en mujeres adultas hipotiroideas el cumplimiento al tratamiento farmacológico según el nivel de conocimiento de la enfermedad, los síntomas y signos que la caracterizan y la forma de controlarla. Material y Métodos: Diseño observacional transversal en muestra no probabilística de mujeres mayores de 40 años con hipotiroidismo primario tratadas con levotiroxina, que asistieron a realizarse prueba de laboratorio a un Instituto de Análisis de la Ciudad Autónoma de Buenos Aires (CABA) entre los meses de agosto y octubre de 2012. Variables: Por interrogatorio directo se estudió el conocimiento de la enfermedad medido por el Test de Batalla y cumplimiento al tratamiento farmacológico medida con el test de Morisky-Green. Estadística con el paquete estadístico SPSS 15.0 estableciendo medidas de tendencia central, Odds Rattio, X² o Prueba de Fisher según el tamaño muestral. Resultados: Se evaluaron 171 mujeres con edad promedio de 54,8 ± 7,2 años. El 57,3 % refiere un correcto conocimiento sobre la enfermedad. El 74,3 % cumple el tratamiento farmacológico. El 97,1 % de la muestra refiere tomar la levotiroxina en ayunas, el 19,9 % olvida alguna vez tomarla y solo el 5,8 % afirma abandonar el fármaco en caso de malestar. Al asociar el conocimiento de la enfermedad con el cumplimiento de la ad­ministración del fármaco, se observó que a pesar que un 42,7 % del total de mujeres no tienen conocimiento sobre la enfermedad, un 29,3 % de ellas igualmente cumple el tratamiento, no encontrándose asociación significativa entre ambas variables (OR = 1,68; IC95 % = 0,84-3,36; p = 0,15). Conclusiones: Poco más de la mitad de la muestra conoce acerca de la enfermedad. La mayoría cumple el tratamiento farmacológico. No se encontró asociación significativa entre el conocimiento de la enfermedad y el cumplimiento de su tratamiento.

Introduction: Non-adherence to treatment may be a cause of therapeutic failure in hypothyroid patients. Aims: To assess adherence to drug treatment in hypothyroid adult women by level of knowledge of the disease, signs and symptoms that characterize it and how to control it. Material and methods: Cross-sectional design; non-random sample of women aged 40 and older, with primary hypothyroidism treated with levothyroxine, who attended the Instituto de Análisis de la Ciudad Autónoma de Buenos Aires (CABA) for laboratory testing between August and October 2012. Variables studied: knowledge of the disease measured by Batalla´s Test and adherence to drug treatment measured by Morinsky Green's Test. Data collection was performed by direct questioning. Statistical analysis performed by SPSS 15.0 establishing measures of central tendency, Odds Ratio X² and Fisher test according to sample size. Results: We evaluated 171 women with an average age of 54.8 ± 7.2 years; 57.3 % reported a correct level of knowledge about the disease, 74.3 % adhered to drug treatment, 97.1 % of the sample reveals taking levothyroxine while fasting, 19.9 % admits sometimes forgetting to take it and only 5.8 % admitted to discontinuing the drug in case of discomfort. When associating knowledge of the disease with adherence to drug administration, we observed that although 42.7 % of women had no knowledge about the disease, 29.3 % of them also adhered to treatment, finding no significant association between the two variables (OR = 1.68; IC95 % = 0.84-3.36; p = 0.15). Conclusions: Just over half of the sample has knowledge about the disease. Most adhere to drug treat­ment. No significant association between knowledge of the disease and adherence to treatment was found.

[Endoscopy versus microsurgery: results in a consecutive series of nonfunctioning pituitary adenomas]. / Résultats comparés de la chirurgie endoscopique et de la microchirurgie dans une série consécutive de macroadénomes hypophysaires non fonctionnels.

Microsurgical removal of nonfunctioning pituitary adenomas (NFPAs) is often subtotal. Removing the blind spots as viewed through the microscope, endoscopic surgery may improve the quality of removal. Our purpose was to compare the results of the two techniques in a series of NFPA patients operated on by a single surgeon. Thirty-six patients with newly diagnosed NFPAs were operated on using a purely endoscopic procedure and 29 with a microsurgical technique. All patients were explored pre- and postoperatively (at 3 and 6 months and then every 12 months) by endocrine assays, ophthalmologic exam, and 3D MRI. The endocrine and ophthalmologic results as well as the quality of resection and the complications from the two techniques were compared. The follow-up duration and the mean tumor volume (higher in the microsurgical group) were the only differences observed between the two groups. Tumor height and the invasion of the cavernous sinus were not different. All patients with preoperative visual impairment in the endoscopic group improved, whereas in the microsurgical group 90.9% improved, 4.5% were stabilized, and 4.5% worsened (p=ns). Regarding anterior pituitary functions, 42.8% of the patients improved in the endoscopic group, 45.7% remained stable, and 11.4% worsened compared to, respectively, 31, 44,8, and 24.1% in the microsurgical group (p=ns). Gross total removal was achieved in 86.1% for the endoscopic group and in only 65.5% for the microsurgical group (p=0.075). Morbidity was similar in the two groups. This retrospective series showed that endoscopic surgery compared to microsurgery increases the quality of NFPA removal with similar morbidity.

[Is there an evidence-based management of idiopathic retroperitoneal fibrosis?]. / Peut-on proposer une prise en charge de la fibrose rétropéritonéale idiopathique validée par les données de la littérature?

BACKGROUND: Nowadays it is quite easy to diagnose idiopathic retroperitoneal fibrosis (IRF), particularly with the help of medical imaging. However there is no guideline about the treatment. PURPOSE: Looking for data about an evidence-based management. METHODS: Screening of the database Medline. Titles and abstracts of articles published between 01/01/1985 and 31/12/2004 have been read to identify clinical trials and series about more than ten patients. RESULTS: No record of any therapeutic trials has been found. Eight series in total, which included 177 patients, were identified. Two of the patients have been treated by an ureteral desobstruction only (endoscopy or nephrostomy), 45 by surgery (ureterolysis), 65 by corticotherapy and 64 both by surgery and steroids. For 38 patients, immunosuppressive drugs were combined with corticotherapy (azathioprine, cyclophosphamide or D-penicillamine). According to the authors, doses and duration of corticotherapy varied. Median follow-up lasted 56 months. The outcome is satisfactory in 73% for surgery alone, 86% for medical treatment alone and 73% for both. The association between steroids therapy and immunosuppressive drugs is efficient in 97% of the cases. No clear data about side effects was mentioned. DISCUSSION: Treatment of the IRF is still empirical, based on surgery and corticotherapy. There is no guideline about the treatment strategy. Although tamoxifen has been proposed, efficacy evidence is lacking. Prospective multicenter studies will help us to progress in the management of the IRF.

Pyrimidinylimidazole inhibitors of p38: cyclic N-1 imidazole substituents enhance p38 kinase inhibition and oral activity.

Optimization of a series of N-1-cycloalkyl-4-aryl-5-(pyrimidin-4-yl)imidazole inhibitors of p38 kinase is reported. Oral administration of inhibitors possessing a cyclohexan-4-ol or piperidin-4-yl group at N-1 in combination with alkoxy, amino(alkyl), phenoxy and anilino substitution at the 2-position of the pyrimidine was found to potently inhibit LPS-induced TNF in mice and rats. The selectivity of these new inhibitors for p38 kinase versus eight other protein kinases is high and in all cases exceeds that of SB 203580.

Phenoxypyrimidine inhibitors of p38alpha kinase: synthesis and statistical evaluation of the p38 inhibitory potencies of a series of 1-(piperidin-4-yl)-4-(4-fluorophenyl)-5-(2-phenoxypyrimidin-4-yl) imidazoles.

As a continuation of our work with 1,4,5 substituted imidazole inhibitors of p38alpha, we report a series of 1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-phenoxy-4-pyrimidinyl) imidazoles related to 7. The compounds have IC50's for inhibition of p38alpha ranging from 6.0 to 650nM. Statistical analysis of the p38beta inhibitor potencies shows a correlation of IC50's with the electron donating strength of low molecular weight substituents.

Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia.

Mitogen-activated protein kinases (MAPKs) are involved in many cellular processes. The stress-activated MAPK, p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Here, we demonstrate focal ischemic stroke-induced p38 enzyme activation (i.e., phosphorylation) in the brain. The second generation p38 MAPK inhibitor SB 239063 was identified to exhibit increased kinase selectivity and improved cellular and in vivo activity profiles, and thus was selected for evaluation in two rat models of permanent focal ischemic stroke. SB 239063 was administered orally pre- and post-stroke and intravenously post-stroke. Plasma concentration levels were achieved in excess of those that effectively inhibit p38 activity. In both moderate and severe stroke, SB 239063 reduced infarct size by 28-41%, and neurological deficits by 25-35%. In addition, neuroprotective plasma concentrations of SB 239063 that reduced p38 activity following stroke also reduced the stroke-induced expression of IL-1beta and TNFalpha (i.e., cytokines known to contribute to stroke-induced brain injury). SB 239063 also provided direct protection of cultured brain tissue to in vitro ischemia. This robust SB 239063-induced neuroprotection emphasizes a significant opportunity for targeting MAPK pathways in ischemic stroke injury, and also suggests that p38 inhibition be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.

SB 239063, a second-generation p38 mitogen-activated protein kinase inhibitor, reduces brain injury and neurological deficits in cerebral focal ischemia.

The stress-activated mitogen-activated protein kinase (MAPK) p38 has been linked to the production of inflammatory cytokines/mediators/inflammation and death/apoptosis following cell stress. In these studies, a second-generation p38 MAPK inhibitor, SB 239063 (IC(50) = 44 nM), was found to exhibit improved kinase selectivity and increased cellular (3-fold) and in vivo (3- to 10-fold) activity over first-generation inhibitors. Oral SB 239063 inhibited lipopolysaccharide-induced plasma tumor necrosis factor production (IC(50) = 2.6 mg/kg) and reduced adjuvant-induced arthritis (51% at 10 mg/kg) in rats. SB 239063 reduced infarct volume (48%) and neurological deficits (42%) when administered orally (15 mg/kg, b.i.d.) before moderate stroke. Intravenous SB 239063 exhibited a clearance of 34 ml/min/kg, a volume of distribution of 3 l/kg, and a plasma half-life of 75 min. An i.v. dosing regimen that provided effective plasma concentrations of 0.38, 0.75, or 1.5 microg/ml (i.e., begun 15 min poststroke and continuing over the initial 6-h p38 activation period) was used. Significant and dose-proportional brain penetration of SB 239063 was demonstrated during these infusion periods. In both moderate and severe stroke, intravenous SB 239063 produced a maximum reduction of infarct size by 41 and 27% and neurological deficits by 35 and 33%, respectively. No effects of the drug were observed on cerebral perfusion, hemodynamics, or body temperature. Direct neuroprotective effects from oxygen and glucose deprivation were also demonstrated in organotypic cultures of rat brain tissue. This robust in vitro and in vivo SB 239063-induced neuroprotection emphasizes the potential role of MAPK pathways in ischemic stroke and also suggests that p38 inhibition warrants further study, including protection in other models of nervous system injury and neurodegeneration.

Saccharomyces cerevisiae Yak1p protein kinase autophosphorylates on tyrosine residues and phosphorylates myelin basic protein on a C-terminal serine residue.

The serine/threonine protein kinase, Yak1p, functions as a negative regulator of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase. In the present work we report that overexpression of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncated form (residues 148-807) lead to growth arrest in PKA compromised yak1 null yeast cells. Both forms of recombinant Yak1p kinase were catalytically active and preferred myelin basic protein (MBP) as a substrate over several other proteins. Phosphopeptide analysis of bovine MBP by tandem MS revealed two major Yak1p phosphorylation sites, Thr-97 and Ser-164. Peptides containing each site were obtained and tested as Yak1p substrates. Both forms of Yak1p phosphorylated a peptide containing the Ser-164 residue with far more efficient kinetics than MBP. The maximal velocity (V(max)) values of the full-length Yak1p reaction were 110+/-21 (Ser-164) and 8.7+/-1.7 (MBP), and those of N-terminally truncated Yak1p were 560.7+/-74.8 (Ser-164) and 34. 4+/-2.2 (MBP) pmol/min per mg of protein. Although neither form of Yak1p was able to phosphorylate two generic protein tyrosine kinase substrates, both were phosphorylated on tyrosine residues in vivo and underwent tyrosine autophosphorylation when reacted with ATP in vitro. Tandem MS showed that Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP. Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak1p phosphotyrosine content and drastically reduced Yak1p activity against exogenous substrates, suggesting that the phosphotyrosine content of the enzyme is essential for its catalytic activity. Although the N-terminally truncated Yak1p was expressed at a lower level than the full-length protein, its catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme. Taken together, our results suggest that Yak1p is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues.

Selective inhibition of phospholipases by atiprimod, a macrophage targeting antiarthritic compound.

Azaspiranes are cationic amphiphilic compounds that are active in a number of models of autoimmune disease and transplantation. Repeated administration of cationic amphiphiles induces phospholipid accumulation in a variety of species. The present study was conducted to explore the mechanism of phospholipid accumulation in rats caused by treatment with the novel azaspirane, SK&F 106615 (atiprimod). Atiprimod inhibited the activities of partially purified phospholipases A(2) and C, but not D, in a noncompetitive manner in vitro. Treatment of rats for 28 days with 10 mg/kg/day of atiprimod increased the contents of arachidonate-containing molecular species within plasmalogen subclasses of hepatic phosphatidylcholine and phosphatidylethanolamine. In contrast, diacyl-linked species were not affected, indicating a selective effect upon an hepatic plasmalogen-selective phospholipase A(2). Taken together, the data suggest that the beneficial effects of atiprimod in autoimmune diseases may involve inhibition of phospholipase A(2) and C activities. Further, the data suggest that atiprimod is a selective inhibitor of plasmalogen-selective phospholipase A(2) in vivo.

p38 mitogen-activated protein kinase inhibitors--mechanisms and therapeutic potentials.

The pyridinylimidazole compounds, exemplified by SB 203580, originally were prepared as inflammatory cytokine synthesis inhibitors. Subsequently, the compounds were found to be selective inhibitors for p38 mitogen-activated protein kinase (MAPK), a member of the MAPK family. SB 203580 inhibits the catalytic activity of p38 MAPK by competitive binding in the ATP pocket. Four homologues of p38 MAPK have been identified to date, and interestingly, their biochemical properties and their respective sensitivities to the inhibitors are distinct. X-ray crystallographic analysis of p38-inhibitor complexes reinforces the observations made from site-directed mutagenesis studies, thereby providing a molecular basis for understanding the kinase selectivity of these inhibitors. The p38 MAPK inhibitors are efficacious in several disease models, including inflammation, arthritis and other joint diseases, septic shock, and myocardial injury.

Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models.

Chronic inflammatory diseases often are accompanied by intense angiogenesis, supporting the destructive proliferation of inflammatory tissues. A model of inflammatory angiogenesis is the murine air pouch granuloma, which has a hyperangiogenic component. In this model, we explored the regulation of inflammatory angiogenesis using SB 220025, a specific inhibitor of human p38 mitogen-activated protein (MAP) kinase, with an IC50 value of 60 nM and 50- to 1000-fold selectivity vs. other kinases tested. In vivo, this compound reduced the lipopolysaccharide-induced production of tumor necrosis factor at an ED50 value of 7.5 mg/kg. In the inflammatory angiogenesis model, over the course of granuloma development, we observed elevated levels of interleukin-1beta and tumor necrosis factor-alpha during the chronic inflammatory phase when intense angiogenesis occurs. SB 220025 at 30 mg/kg b.i.d. p.o. was able to greatly reduce the expression of these cytokines and inhibit angiogenesis by approximately 40%. To further study the effects of p38/CSBP MAP kinase inhibition in angiogenesis-dependent chronic inflammatory disease, SB 220025 was tested in murine collagen-induced arthritis. In this model, SB 220025 was able to prevent the progression of established arthritis. Thus, this p38/CSBP MAP kinase inhibitor, which can reduce inflammatory cytokine production and inhibit angiogenesis, is an effective treatment for chronic proliferative inflammatory disease.

Pyrimidinylimidazole inhibitors of CSBP/p38 kinase demonstrating decreased inhibition of hepatic cytochrome P450 enzymes.

Pyrimidine analogs of the pyrimidinylimidazole class of CSBP/p38 kinase inhibitors were prepared in an effort to reduce the potent inhibition of hepatic cytochrome P450 observed for the pyridinyl compounds. The substitution of pyrimidin-4-yl, 2-methoxypyrimidin-4-yl, or 2-methylaminopyrimidin-4-yl for pyridin-4-yl effectively dissociates CSBP/p38 kinase from P450 inhibition for this series and furthermore achieves an increase in oral activity.

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site.

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

Regulation of stress-induced cytokine production by pyridinylimidazoles; inhibition of CSBP kinase.

Members of three classes of pyridinylimidazoles bind with varying affinities to CSBP (p38) kinase which is a member of a stress-induced signal transduction pathway. Based upon SAR and protein homology modeling, the pharmacophore and three potential modes of binding to the enzyme are presented. For a subset of pyridinylimidazoles, binding is shown to correlate with inhibition of CSBP kinase activity, whereas no significant inhibition of PKA, PKC alpha and ERK kinase activity is observed.

Protein kinase C regulates TNF-alpha production by human monocytes.

Tumor Necrosis Factor alpha (TNF alpha) is a cytokine mediator that is produced primarily by activated monocytes/macrophages in response to endotoxin/lipopolysaccharide (LPS) as well as other stimuli. The second messenger systems that regulate the synthesis and release of TNF alpha are not clearly defined. In the present study, the role of protein kinase C (PKC) in the production of TNF alpha was investigated in human peripheral blood monocytes stimulated with either LPS or zymosan. Two broad spectrum protein kinase inhibitors (staurosporine and K252a) and two PKC specific inhibitors (calphostin C and chelerythrine), were used as probes to delineate the involvement of PKC in the production of TNF alpha. The results indicate that inhibition of PKC diminished LPS- or zymosan- induced TNF alpha production in a concentration-dependent manner. The IC50 values for the inhibition of TNF alpha production were 0.2 nM for staurosporine, and 20 nM for K252a, Calphostin C and chelerythrine. Furthermore, long term PMA treatment of these cells (to abrogate PKC-mediated responses) resulted in a significant reduction of stimuli-induced TNF alpha production. LPS and zymosan also induced an increase in membrane associated PKC activity in human monocytes, which could be inhibited by pretreatment of the cells with calphostin C. Finally, western blot analysis with PKC isoform-specific antibodies demonstrates that the alpha and xi are the predominent isoforms expressed in human monocytes. These data strongly suggest that an initial step in TNF alpha production by human monocytes challenged with physiological stimulants, such as LPS and zymosan, involves a PKC-dependent mechanism.

Inhibition of interleukin-1 (IL-1) and tumor necrosis factor (TNF) production by pyridinyl imidazole compounds is independent of cAMP elevating mechanisms.

Exposure of human monocytes (HM) to E. coli lipopolysaccharide (LPS) results in measurable production of both IL-1 beta and TNF alpha in culture supernatants. It has previously been reported that the elevation of cAMP levels in HM selectively suppresses the LPS-induced TNF alpha but not IL-1 beta production. In this study we investigated whether the novel anti-inflammatory drug, SK&F 86002 [5-4(-pyridyl)-6(4-fluorophenyl)-2,3-dihydroimidazole(2,1-b)thi azol] and related analogs of the pyridinyl imidazole class, inhibit IL-1 and TNF production via a cAMP-dependent mechanism. These compounds, when added together with LPS result in inhibition of IL-1 and TNF production with equal-rank-order potency. Although the pyridinyl imidazole compounds were found to be generally weak phosphodiesterase inhibitors, they did not affect cAMP levels in HM, alone or in the presence of LPS. In contrast, PGE2, which significantly elevated intracellular cAMP levels, inhibited TNF but not IL-1 production at the transcriptional level. Taken together, these results suggest that the pyridinyl imidazoles inhibit the production of IL-1 beta and TNF alpha through pathways independent of cAMP elevating mechanisms.

Effect of human immunodeficiency virus gp120 glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human T lymphocytes.

The human immunodeficiency virus binds to CD4+ T lymphocytes through the interaction of its envelope glycoprotein (gp120) with the CD4 molecule. The src-related protein tyrosine kinase p56lck is physically associated with CD4 and is co-immunoprecipitated by CD4 monoclonal antibody (mAb). Activators of protein kinase C (PKC) cause the dissociation of p56lck from CD4. Here we report that gp120 mAb immunoprecipitated the p56lck.CD4.gp120 complex after short term treatment (20 min) of human T lymphocytes with gp120. The p56lck that was associated with the CD4.gp120 complex was dissociated by activators of PKC. This effect was abolished by pretreatment of cells with PKC inhibitors. Thus the p56lck.CD4.gp120 immune complex immunoprecipitated by gp120 mAb behaves in a similar manner, with respect to PKC activation or inhibition, to the p56lck.CD4 complex immunoprecipitated by CD4 mAb. Short term treatment of cells with gp120, followed by gp120 mAb, resulted in an increase in the tyrosine kinase activity of p56lck associated with CD4. However, the amount of enzyme associated with CD4 remained unchanged. Long term treatment (20 h) of human T lymphocytes with gp120 resulted in the down-regulation of cell surface CD4 molecules. A parallel decrease in CD4-associated gp120 was also observed. In addition, gp120 caused the dissociation of p56lck and CD4. However, the dissociation of the p56lck from CD4 occurred at much faster rate than the down-regulation of surface CD4 molecules. Such mechanisms may account for the down-regulation of cell surface CD4 molecules and the depletion of functional CD4+ T lymphocytes which are characteristic of human immunodeficiency virus infections and acquired immune deficiency syndrome pathogenesis.

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C'' and FG. This structure is consistent with C'C'' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.

Effects of prostaglandins and cAMP levels on monocyte IL-1 production.

The effects of prostaglandin E2 (PGE2), as well as other cAMP-elevating agents, on the in vitro production of interleukin-1 (IL-1) by human monocytes (HM) were examined. Exposure to E. coli lipopolysacharide (LPS) resulted in a dose- and time-dependent increase of IL-1 activity in monocytes culture supernatants. Maximal levels of secreted IL-1 in response to 10 ng LPS/ml were obtained at 18 h. PGE1, PGE2, cholera toxin (CT) and the phosphodiesterase inhibitor, isobutylmethylxanthin (IBMX), when added with LPS, resulted in a dose-dependent increase in cellular cAMP and in secreted IL-1. Maximal levels of secreted IL-1 were 2.5-5.0-fold over LPS alone. When CT or PGE2 was added with IBMX a further increase was observed. These agents exhibited marginal effect on cell-associated IL-1. Maximum cAMP levels was achieved at 10 min in response to either PGE1 or PGE2 and returned to near basal levels after 18 h. While PGE1 elevated cAMP to a larger extent than PGE2 (58- vs. 30-fold) the latter resulted in a higher levels of secreted IL-1. Elevated cAMP persisted throughout the entire culture period in response to CT (4-6-fold) or IBMX (7-fold). Thus, we conclude that in adherent HM, IL-1 production is potentiated and not inhibited by prostaglandins or agents that elevate cellular cAMP.