Characterization of apxIVA, a new RTX determinant of Actinobacillus pleuropneumoniae

A fourth type of RTX determinant was identified in Actinobacillus pleuropneumoniae and was designated apxIVA. When expressed in Escherichia coli, recombinant ApxIVA showed a weak haemolytic activity and co-haemolytic synergy with the sphingomyelinase (beta-toxin) of Staphylococcus aureus. These activities required the presence of an additional gene, ORF1, that is located immediately upstream of apxIVA. The apxIVA gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with A. pleuropneumoniae serotypes 1, 5 and 7 started to produce antibodies that reacted with recombinant ApxIVA 14 d post-infection, indicating that apxIVA is expressed in vivo. In addition, sera from pigs naturally and experimentally infected with any of the serotypes all reacted with recombinant ApxIVA. The apxIVA gene from the serotype 1 A. pleuropneumoniae type strain Shope 4074T encodes a protein with a predicted molecular mass of 202 kDa which has typical features of RTX proteins including hydrophobic domains in the N-terminal half and 24 glycine-rich nonapeptides in the C-terminal half that bind Ca2+. The glycine-rich nonapeptides are arranged in a modular structure and there is some variability in the number of modules in the ApxIVA proteins of different serotypes of A. pleuropneumoniae. The deduced amino acid sequences of the ApxIVA proteins have significant similarity with the Neisseria meningitidis iron-regulated RTX proteins FrpA and FrpC, and to a much lesser extent with other RTX proteins. The apxIVA gene could be detected in all A. pleuropneumoniae serotypes and seems to be species-specific. Although the precise role of this new RTX determinant in pathogenesis of porcine pleuropneumonia needs to be determined, apxIVA is the first in vivo induced toxin gene that has been described in A. pleuropneumoniae.

Parvovirus B19 uptake is a highly selective process controlled by VP1u, a novel determinant of viral tropism

The VP1 unique region (VP1u) of human parvovirus B19 (B19V) is the immunodominant part of the viral capsid. Originally inaccessible, the VP1u becomes exposed upon primary attachment to the globoside receptor. To study the function of the exposed VP1u in B19V uptake, we expressed this region as a recombinant protein. Here, we report that purified recombinant VP1u binds and is internalized in UT7/Epo cells. By means of truncations and specific antibodies, we identified the most N-terminal amino acid residues of VP1u as the essential region for binding and internalization. Furthermore, the recombinant VP1u was able to block B19V uptake, suggesting that the protein and the virus undertake the same internalization pathway. Assays with different erythroid and nonerythroid cell lines showed that the N-terminal VP1u binding was restricted to a few cell lines of the erythroid lineage, which were also the only cells that allowed B19V internalization and infection. These results together indicate that the N-terminal region of VP1u is responsible for the internalization of the virus and that the interacting receptor is restricted to B19V-susceptible cells. The highly selective uptake mechanism represents a novel determinant of the tropism and pathogenesis of B19V.

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection.

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F(WT)) and attachment (H(WT)) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H(WT) determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F(WT) reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

Planning and implementation efficacy as a determinant of health risk communication outcomes: Case studies of the U.S. Public Health Service (PHS)

This study was conducted under the auspices of the Subcommittee on Risk Communication and Education of the Committee to Coordinate Environmental Health and Related Programs (CCEHRP) to determine how Public Health Service (PHS) agencies are communicating information about health risk, what factors contributed to effective communication efforts, and what specific principles, strategies, and practices best promote more effective health risk communication outcomes.^ Member agencies of the Subcommittee submitted examples of health risk communication activities or decisions they perceived to be effective and some examples of cases they thought had not been as effective as desired. Of the 10 case studies received, 7 were submitted as examples of effective health risk communication, and 3, as examples of less effective communication.^ Information contained in the 10 case studies describing the respective agencies' health risk communication strategies and practices was compared with EPA's Seven Cardinal Rules of Risk Communication, since similar rules were not found in any PHS agency. EPA's rules are: (1) Accept and involve the public as a legitimate partner. (2) Plan carefully and evaluate your efforts. (3) Listen to the public's specific concerns. (4) Be honest, frank, and open. (5) Coordinate and collaborate with other credible sources. (6) Meet the needs of the media. (7) Speak clearly and with compassion.^ On the basis of case studies analysis, the Subcommittee, in their attempts to design and implement effective health risk communication campaigns, identified a number of areas for improvement among the agencies. First, PHS agencies should consider developing a focus specific to health risk communication (i.e., office or specialty resource). Second, create a set of generally accepted practices and guidelines for effective implementation and evaluation of PHS health risk communication activities and products. Third, organize interagency initiatives aimed at increasing awareness and visibility of health risk communication issues and trends within and between PHS agencies.^ PHS agencies identified some specific implementation strategies the CCEHRP might consider pursuing to address the major recommendations. Implementation strategies common to PHS agencies emerged in the following five areas: (1) program development, (2) building partnerships, (3) developing training, (4) expanding information technologies, and (5) conducting research and evaluation. ^

Modulation of BCL-X$\sb{\rm L}$ is a critical determinant of c-myc induced apoptosis

The fine balance between proliferation and apoptosis plays a primary role in carcinogenesis. Proto-oncogenes that induce both proliferation and apoptosis provide a powerful inbuilt system to inhibit clonal expansion of cells with high proliferation rates. This provides a restraint to the development of neoplasms. C-myc expressing cells undergo apoptosis in low serum by an unknown mechanism. Several lines of evidence suggested that c-myc induces apoptosis by a transcriptional mechanism. However, the target genes of this program have not been fully defined. Protein synthesis inhibitors induce apoptosis in c-myc over-expressing cells at high serum levels suggesting that inhibition of synthesis of a survival factor may induce apoptosis. We show that the expression of c-myc directly correlates with an increase in the level of a survival protein, bcl-$\rm x\sb{L},$ and a decrease in the pro-apoptotic protein, bax, at both the protein and mRNA level. Furthermore, a significant decrease of the bcl-$\rm x\sb{L}$ protein levels is observed under low serum conditions. In order to investigate the mechanism of regulation of bcl-$\rm x\sb{L}$ and bax by c-myc, the bcl-x and bax promoters were cloned, sequenced and shown to contain c-myc binding sites. The chloramephenicol acetyl transferase (CAT) reporter assay was used to demonstrate activation of the bcl-x promoter by increasing levels of c-myc when co-transfected in COS cells. The bax promoter was also shown to be transrepressed in c-myc expressing cells. The role of bcl-$\rm x\sb{L}$ in apoptosis regulation in c-myc cell lines in normal and low serum was then investigated. Cells lines expressing c-myc and bcl-$\rm x\sb{L}$ were generated and were shown to be resistant to apoptosis induction in low serum. Furthermore, cell lines expressing c-myc, anti-sense bcl-$\rm x\sb{L}$ and $\beta$-galactosidase demonstrated significantly enhanced rates of apoptosis in high serum compared to c-myc Rat 1a cells. These findings suggest that c-myc activates a survival program involving bcl-$\rm x\sb{L}$ upregulation and bax downregulation. However, this survival signal is reduced under low serum conditions by the relative downregulation of bcl-$\rm x\sb{L}$ allowing for apoptosis to proceed. These data also directly demonstrates that downregulation in the level of bcl-$\rm x\sb{L}$ associated with low serum conditions is a critical determinant of c-myc induced apoptosis. ^

Mechanism of translationally coupled mRNA turnover mediated by c-fos protein-coding-region determinant

Proto-oncogene c-fos is a member of the class of early-response genes whose transient expression plays a crucial role in cell proliferation, differentiation, and apoptosis. Degradation of c- fos mRNA is an important mechanism for controlling c-fos expression. Rapid mRNA turnover mediated by the protein-coding-region determinant (mCRD) of the c-fos transcript illustrates a functional interplay between mRNA turnover and translation that coordinately influences the fate of cytoplasmic mRNA. It is suggested that mCRD communicates with the 3′ poly(A) tail via an mRNP complex comprising mCRD-associated proteins, which prevents deadenylation in the absence of translation. Ribosome transit as a result of translation is required to alter the conformation of the mRNP complex, thereby eliciting accelerated deadenylation and mRNA decay. To gain further insight into the mechanism of mCRD-mediated mRNA turnover, Unr was identified as an mCRD-binding protein, and its binding site within mCRD was characterized. Moreover, the functional role for Unr in mRNA decay was demonstrated. The result showed that elevation of Unr protein level in the cytoplasm led to inhibition of mRNA destabilization by mCRD. In addition, GST pull-down assay and immuno-precipitation analysis revealed that Unr interacted with PABP in an RNA-independent manner, which identified Unr as a novel PABP-interacting protein. Furthermore, the Unr interacting domain in PABP was characterized. In vivo mRNA decay experiments demonstrated a role for Unr-PABP interaction in mCRD-mediated mRNA decay. In conclusion, the findings of this study provide the first evidence that Unr plays a key role in mCRD-mediated mRNA decay. It is proposed that Unr is recruited by mCRD to initiate the formation of a dynamic mRNP complex for communicating with poly(A) tail through PABP. This unique mRNP complex may couple translation to mRNA decay, and perhaps to recruit the responsible nuclease for deadenylation. ^

Determinant factors to discriminate the body weight loss in overweight people

Introduction. Most studies have described how the weight loss is when different treatments are compared (1-3), while others have also compared the weight loss by sex (4), or have taken into account psychosocial (5) and lifestyle (6, 7) variables. However, no studies have examined the interaction of different variables and the importance of them in the weight loss. Objective. Create a model to discriminate the range of weight loss, determining the importance of each variable. Methods. 89 overweight people (BMI: 25-29.9 kg?m-2), aged from 18 to 50 years, participated in the study. Four types of treatments were randomly assigned: strength training (S), endurance training (E), strength and endurance training (SE), and control group (C). All participants followed a 25% calorie restriction diet. Two multivariate discriminant models including the variables age, sex, height, daily energy expenditure (EE), type of treatment (T), caloric restriction (CR), initial body weight (BW), initial fat mass (FM), initial muscle mass (MM) and initial bone mineral density (BMD) were performed having into account two groups: the first and fourth quartile of the % of weight loss in the first model; the groups above and below the mean of the % of weight loss in the second model. The discriminant models were built using the inclusion method in SPSS allowing us to find a function that could predict the body weight loss range that an overweight person could achieve in a 6 months weight loss intervention.Results. The first discriminant analysis predicted that a combination of the studied variables would discriminate between the two ranges of body weight loss with 81.4% of correct classification. The discriminant function obtained was (Wilks? Lambda=0.475, p=0.003): Discriminant score=-18.266-(0.060xage)- (1.282xsex[0=female;1=male])+(14.701xheight)+(0.002xEE)- (0.006xT[1=S;2=E;3=SE;4=C])-(0.047xCR)- (0.558xBW)+(0.475xFM)+(0.398xMM)+(3.499xBMD) The second discriminant model obtained would discriminate between the two groups of body weight loss with 74.4% of correct classification. The discriminant function obtained was (Wilks? Lambda=0.725, p=0.005): Discriminant score=-5.021-(0.052xage)- (0.543xsex[0=female;1=male])+(3.530xheight)+(0.001xEE)- (0.493xT[1=S;2=E;3=SE;4=C])+(0.003xCR)- (0.365xBW)+(0.368xFM)+(0.296xMM)+(4.034xBMD) Conclusion. The first developed model could predict the percentage of weight loss in the following way: if the discriminant score is close to 1.051, the range of weight loss will be from 7.44 to -4.64% and if it is close to - 1.003, the range will be from -11.03 to -25,00% of the initial body weight. With the second model if the discriminant score is close to 0.623 the body weight loss will be above -7.93% and if it is close to -0.595 will be below - 7.93% of the initial body weight. References. 1. Brochu M, et al. Resistance training does not contribute to improving the metabolic profile after a 6-month weight loss program in overweight and obese postmenopausal women. J Clin Endocrinol Metab. 2009 Sep;94(9):3226-33. 2. Del Corral P, et al. Effect of dietary adherence with or without exercise on weight loss: a mechanistic approach to a global problem. J Clin Endocrinol Metab. 2009 May;94(5):1602-7. 3. Larson-Meyer DE, et al. Caloric Restriction with or without Exercise: The Fitness vs. Fatness Debate. Med Sci Sports Exerc. 2010;42(1):152-9. 4. Hagan RD, et al. The effects of aerobic conditioning and/or caloric restriction in overweight men and women. Medicine & Science in Sports & Exercise. 1986;18(1):87-94. 5. Teixeira PJ, et al. Mediators of weight loss and weight loss maintenance in middle-aged women. Obesity (Silver Spring). 2010 Apr;18(4):725-35. 6. Bautista-Castano I, et al. Variables predictive of adherence to diet and physical activity recommendations in the treatment of obesity and overweight, in a group of Spanish subjects. Int J Obes Relat Metab Disord. 2004 May;28(5):697-705.

El fitness como factor determinante en el riesgo de discapacidad, su relación con la composición corporal y el estado cognitivo en población mayor institucionalizada y de vida independiente = Fitness as a determinant factor in the risk of disability, and its relationship to body composition and cognitive status in institutionalized elderly population and independent living

La condición física, o como mejor se la conoce hoy en día el “fitness”, es una variable que está cobrando gran protagonismo, especialmente desde la perspectiva de la salud. La mejora de la calidad de vida que se ha experimentado en los últimos años en las sociedades desarrolladas, conlleva un aumento de la esperanza de vida, lo que hace que cada vez más personas vivan más años. Este rápido crecimiento de la población mayor de 60 años hace que, un grupo poblacional prácticamente olvidado desde el punto de vista de la investigación científica en el campo de la actividad física y del deporte, cobre gran relevancia, con el fin de poder ayudar a alcanzar el dicho “no se trata de aportar años a la vida sino vida a lo años”. La presente memoria de Tesis Doctoral tiene como principal objetivo valorar los niveles de fitness en población mayor española, además de analizar la relación existente entre el fitness, sus condicionantes y otros aspectos de la salud, tales como la composición corporal y el estado cognitivo. Entendemos que para poder establecer futuras políticas de salud pública en relación a la actividad física y el envejecimiento activo es necesario conocer cuáles son los niveles de partida de la población mayor en España y sus condicionantes. El trabajo está basado en los datos del estudio multicéntrico EXERNET (Estudio Multi-céntrico para la Evaluación de los Niveles de Condición Física y su relación con Estilos de Vida Saludables en población mayor española no institucionalizada), así como en los datos de dos estudios, llevados a cabo en población mayor institucionalizada. Se han analizado un total de 3136 mayores de vida independiente, procedentes de 6 comunidades autónomas, y 153 mayores institucionalizados en residencias de la Comunidad de Madrid. Los principales resultados de esta tesis son los siguientes: a) Fueron establecidos los valores de referencia, así como las curvas de percentiles, para cada uno de los test de fitness, de acuerdo a la edad y al sexo, en población mayor española de vida independiente y no institucionalizada. b) Los varones obtuvieron mejores niveles de fitness que las mujeres, excepto en los test de flexibilidad; existe una tendencia a disminuir la condición física en ambos sexos a medida que la edad aumenta. c) Niveles bajos de fitness funcional fueron asociados con un aumento en la percepción de problemas. d) El nivel mínimo de fitness funcional a partir del cual los mayores perciben problemas en sus actividades de la vida diaria (AVD) es similar en ambos sexos. e) Niveles elevados de fitness fueron asociados con un menor riesgo de sufrir obesidad sarcopénica y con una mejor salud percibida en los mayores. f) Las personas mayores con obesidad sarcopénica tienen menor capacidad funcional que las personas mayores sanas. g) Niveles elevados de fuerza fueron asociados con un mejor estado cognitivo siendo el estado cognitivo la variable que más influye en el deterioro de la fuerza, incluso más que el sexo y la edad. ABSTRACT Fitness is a variable that is gaining in prominence, especially from the health perspective. Improvement of life quality that has been experienced in the last few years in developed countries, leads to an expanded life expectancy, increasing the numbers of people living longer. This population consisting of people of over 60 years, an almost forgotten population group from the point of view of scientific research in the field of physical activity and sport, is becoming increasingly important, with the main aim of helping to achieve the saying “do not only add years to life, but also add life to years”. The principal aim of the current thesis was to assess physical fitness levels in Spanish elderly people, of over 65 years, analyzing relationship between physical fitness, its determinants, and other aspects of health such as body composition and cognitive status. In order to establish further public health policies in relation to physical activity and active ageing it is necessary to identify the starting physical fitness levels of the Spanish population and their determinants. The work is based on data from the EXERNET multi-center study ("Multi-center Study for the Evaluation of Fitness levels and their relationship to Healthy Lifestyles in noninstitutionalized Spanish elderly"), and on data from two studies conducted in institutionalized elderly people: a total of 3136 non-institutionalized elderly, from 6 Regions of Spain, and 153 institutionalized elderly in nursing homes of Madrid. The main outcomes of this thesis are: a) sex- and age-specific physical fitness normative values and percentile curves for independent and non-institutionalized Spanish elderly were established. b) Greater physical fitness was present in the elderly men than in women, except for the flexibility test, and a trend toward decreased physical fitness in both sexes as their age increased. c) Lower levels of functional fitness were associated with increased perceived problems. d) The minimum functional fitness level at which older adults perceive problems in their ADLs, is similar for both sexes e) Higher levels of physical fitness were associated with a reduced risk of suffering sarcopenic obesity and better perceived health among the elderly. f) The elderly with sarcopenic obesity have lower physical functioning than healthy counterparts. g) Higher strength values were associated with better cognitive status with cognitive status being the most influencing variable in strength deterioration even more than sex and age.

Fuel poverty as a determinant in energy retrofitting actions

Fuel poverty can be defined as “the inability to afford adequate warmth in the home" and it is the result of the combination of three items: low household income, housing lack of energy efficiency and high energy bills. Although it affects a growing number of households within the European Union only some countries have an official definition for it. In 2013, the European Parliament claimed the Commission and Estate Members to develop different policies in order to fight household energy vulnerability. The importance of tackling fuel poverty is based on the critical consequences it has for human health living below certain temperatures. In Spain some advances have been made in this field but main existing studies remain at the statistical level and do not deepen the understanding of the problem from the perspective of dwelling indoor habitability conditions. What is more, this concept is yet to be officially defined. This paper presents the evaluation of fuel poverty in a building block of social housing located in the centre of Zaragoza and how this issue determined the strategies implemented in the energy retrofitting intervention project. At a first step, fuel poverty was appraised through the exploration of indoor thermal conditions. The adaptive thermal comfort (UNE-EN 15251:2008) method was used to establish the appropriate indoor temperatures and consequently to determine what can be called 'comfort gap'. Results were collated and verified with energy bills collection and a survey work that gathered data from neighbours. All this permitted pointing out those households more in need. Results from the social analysis combined with the evaluation of the building thermal performance determined the intervention. The renovation project was aimed at the implementation of passive strategies that improve households thermal comfort in order to alleviate households fuel poverty situation. This research is part of the project NewSolutions4OldHousing (LIFE10 ENV/ES/439) cofounded by the European Commission under the LIFE+ Programme.

The anterior determinant bicoid of Drosophila is a derived Hox class 3 gene

The Drosophila gene bicoid functions as the anterior body pattern organizer of Drosophila. Embryos lacking maternally expressed bicoid fail to develop anterior segments including head and thorax. In wild-type eggs, bicoid mRNA is localized in the anterior pole region and the bicoid protein forms an anterior-to-posterior concentration gradient. bicoid activity is required for transcriptional activation of zygotic segmentation genes and the translational suppression of uniformly distributed maternal caudal mRNA in the anterior region of the embryo. caudal genes as well as other homeobox genes or members of the Drosophila segmentation gene cascade have been found to be conserved in animal evolution. In contrast, bicoid homologs have been identified only in close relatives of the schizophoran fly Drosophila. This poses the question of how the bicoid gene evolved and adopted its unique function in organizing anterior–posterior polarity. We have cloned bicoid from a basal cyclorrhaphan fly, Megaselia abdita (Phoridae, Aschiza), and show that the gene originated from a recent duplication of the direct homolog of the vertebrate gene Hox3, termed zerknüllt, which specifies extraembryonic tissues in insects.

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

A secreted Salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria

Bacterial pathogens have evolved sophisticated mechanisms to interact with their hosts. A specialized type III protein secretion system capable of translocating bacterial proteins into host cells has emerged as a central factor in the interaction between a variety of mammalian and plant pathogenic bacteria with their hosts. Here we describe AvrA, a novel target of the centisome 63 type III protein secretion system of Salmonella enterica. AvrA shares sequence similarity with YopJ of the animal pathogen Yersinia pseudotuberculosis and AvrRxv of the plant pathogen Xanthomonas campestris pv. vesicatoria. These proteins are the first examples of putative targets of type III secretion systems in animal and plant pathogenic bacteria that share sequence similarity. They may therefore constitute a novel family of effector proteins with related functions in the cross-talk of these pathogens with their hosts.

Endoplasmic reticulum quality control of asialoglycoprotein receptor H2a involves a determinant for retention and not retrieval

The human asialoglycoprotein receptor H2a subunit contains a charged pentapeptide, EGHRG, in its ectodomain that is the only sequence absent from the H2b alternatively spliced variant. H2b exits the endoplasmic reticulum (ER) even when singly expressed, whereas H2a gives rise to a cleaved soluble secreted ectodomain fragment; uncleaved membrane-bound H2a molecules are completely retained and degraded in the ER. We have inserted the H2a pentapeptide into the sequence of the H1 subunit (H1i5), which caused complete ER retention but, unexpectedly, no degradation. This suggests that the pentapeptide is a determinant for ER retention not colocalizing in H2a with the determinant for degradation. The state of sugar chain processing and the ER localization of H1i5, which was unchanged at 15°C or after treatment with nocodazole, indicate ER retention and not retrieval from the cis-Golgi or the intermediate compartment. H1i5 folded similarly to H1, and both associated to calnexin. However, whereas H1 dissociated with a half time of 45 min, H1i5 remained bound to the chaperone for prolonged periods. The correct global folding of H2a and H1i5 and of other normal precursors and unassembled proteins and the true ER retention, and not exit and retrieval, suggest a difference in their quality control mechanism compared with that of misfolded proteins, which does involve retrieval. However, both pathways may involve calnexin.

GPIIIa-(49–66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia

High-affinity (Kd = 1 × 10−9 M) anti-platelet GPIIIa has been isolated from serum immune complexes of immunologic thrombocytopenic HIV-1-infected patients (HIV-1-ITP). Affinity-purified anti-platelet antibody reacted with a recombinant GPIIIa-(1–200) and -(1–66) fusion peptide and with an 18-mer GPIIIa-(49–66) peptide but not with seven other GPIIIa peptides spanning the length of GPIIIa. Most of the anti-platelet antibody (≈85%) could be adsorbed to and eluted from a GPIIIa-(49–66) affinity column. Binding of antibody to platelets could be inhibited by GPIIIa-(49–66) or an equimolar peptide-albumin conjugate (IC50 = 2 μM). Sera from 7 control subjects and 10 classic autoimmune thrombocytopenic patients gave background reactivity with GPIIIa-(49–66). HIV-1-ITP sera from 16 patients reacted with a mean OD 6-fold greater than background (range, 4- to 9-fold). Serum anti-GPIIIa-(49–66) concentration correlated inversely with platelet count, R2 = 0.51, n = 31, P < 0.0001. Because mouse platelet GPIIIa-(49–66) has 83% homology with human GPIIIa and mouse monocytes contain Fc receptors for the human IgG1-κ/λ antibody, we determined the in vivo effect of human anti-GPIIIa on mouse platelets. Affinity-purified antibody, 25–50 μg given i.p., resulted in a precipitous drop in platelet count to 30% of baseline, with nadir at 4 hr and return to normal in 36 hr. No effect was noted with control IgG. Acute thrombocytopenia could be prevented or reversed by the injection of the GPIIIa-(49–66) albumin conjugate at zero time or 2 hr after antibody, respectively, but not with a scrambled peptide-albumin conjugate. Thus HIV-1-ITP patients have high-affinity anti-platelet GPIIIa against a major antigenic determinant, GPIIIa-(49–66), which correlates inversely with platelet count and induces thrombocytopenia in mice.