Utility of Allograft Talus as a Source for Grafting of Concurrent Humeral and Glenoid Defects Associated With Anterior Glenohumeral Instability: An Anthropometric Analysis.

PURPOSE: To compare morphologic data of the talus using magnetic resonance images with previously reported values of the humeral head and the glenoid as a potential graft source for both the humeral head as well as glenoid reconstruction in the setting of concomitant glenoid and humeral head defects. METHODS: All magnetic resonance images of the ankle were reviewed for assessment of the morphology and variation of the talus among individuals. Patients with post-traumatic, osteoarthritic, or surgical changes to the distal tibia about the mid- or hind- foot, or patients with incomplete medical records were excluded. Radiographic parameters that were measured included the maximum vertical height (MVH), the height to the talar neck, the radius of curvature (ROC) of the talar dome, ROC of the subtalar joint, and the maximum medial-to-lateral width of the talar dome. Demographic data also were collected on each individual. Statistical analysis was performed via a linear regression model with backwards elimination to determine which demographic data correlated most strongly with talar anthropometric values. RESULTS: A total of 82 study patients met inclusion criteria (59 male, 23 female; mean age 40.91 ± 14.69 years). Sex was found have a positive correlation of the following talar dimensions: MVH (P = .039), talar dome ROC (P < .001), and subtalar joint ROC (p = 0.001). Height was the most positive correlation for medial-to-lateral width (P < .001), height to the talar neck (P = .004), and also correlate for MVH (P = .004). Body mass index was found to have multicollinearity and was therefore not used as a variable. CONCLUSIONS: Allograft talus appears to be a viable graft, as demonstrated in this anthropometric study for both reconstruction of the glenoid and humeral head when cases of bipolar glenohumeral bone loss are present. CLINICAL RELEVANCE: This study aims to further evaluate potential allograft donor sites for bipolar lesions.

Use of the Contralateral Glenoid for Calculation of Glenoid Bone Loss: A Cadaveric Anthropometric Study.

PURPOSE: The purpose of this study was to determine if there are significant side-to-side anthropometric differences between paired glenoids. METHODS: Forty-six matched-pair cadaver glenoids were harvested, and their glenoid heights (GHs) and glenoid widths (GWs) were measured with digital calipers. The glenoid surface area was calculated using the standard assumption that the inferior two-thirds of the glenoid is a perfect circle. RESULTS: There was a statistically significant difference between matched-pair GHs of 0.96 ± 3.07 mm (P = .020) and GWs of 0.46 ± 1.64 mm (P = .033). There was a significant difference of glenoid cavity area of 20.30 ± 81.53 mm2 (P = .044), or a difference of ∼3%. A total of 4 of 46 pairs of glenoids (8.6%) showed a difference in width >3 mm. CONCLUSIONS: This study demonstrates the fallacy of use of the contralateral glenoid in measuring glenoid bone loss. Although many paired samples exhibited similar side-to-side glenoid measurements, the number of cadaveric pairs that showed differences of >3 mm was substantial. Caution should be taken when using calculation methods that include this assumption for surgical decision making, as surface area, GW, and GH were all shown to have statistically significant side-to-side differences in their measurements. CLINICAL RELEVANCE: Many methods exist for measuring glenoid bone loss after anterior shoulder dislocation, but some of the current methods may be inaccurate and lead to unreliable estimations.

Towards community-based in situ conservation strategies: a typological analysis of Borgou cattle herding systems in northeastern Benin.

A thorough knowledge of cattle herding systems is very important for planning sustainable genetic improvement and conservation strategies. This paper is initiated to characterize Benin native Borgou cattle farming systems in its department of origin by mean of survey including 180 cattle farmers owning at least one phenotype of that breed. Using multiple correspondence analysis and hierarchical ascending classification, four groups of Borgou cattle farms have been identified. The first group qualified as "semi-intensive purebred Borgou cattle farming" own high numbers (87.2 ± 3.95 heads) purchased and reared with the view to promote its genetic improvement, its production, and its conservation as Benin animal genetic resource. The second group (sedentary purebred Borgou cattle farming) is represented by Bariba ethnic group with small numbers (22.18 ± 0.71 heads) of purebred Borgou cattle used mainly for draught. Cattle farmers of the third group are "large transhumant of Zebu and Borgou crossbred cattle farmers" represented by Fulani and Gando ethnic groups whose herds are generally composed of high numbers (75.20 ± 3.43 heads) of cattle acquired by purchasing, inheriting, and fostering. The last one is the "small transhumant of Zebu and Borgou crossbred cattle farming" with an average herd size of 31.98 ± 0.72 heads. Cattle farming is their main activity and animals are used for the production of milk and cheese. These distinctions between Borgou cattle farmers can be an anchorage point for designing sustainable community-based in situ conservation strategies for safeguarding this local breed in its original cradle.

Inhaled PYY(3-36) dry-powder formulation for appetite suppression.

OBJECTIVE: Peptide YY3-36 [PYY(3-36)] has shown efficacy in appetite suppression when dosed by injection modalities (intraperitoneal (IP)/subcutaneous). Transitioning to needle-free delivery, towards inhalation, often utilizes systemic pharmacokinetics as a key endpoint to compare different delivery methods and doses. Systemic pharmacokinetics were evaluated for PYY3-36 when delivered by IP, subcutaneous, and inhalation, the systemic pharmacokinetics were then used to select doses in an appetite suppression pharmacodynamic study. METHODS: Dry-powder formulations were manufactured by spray drying and delivered to mice via nose only inhalation. The systemic plasma, lung tissue, and bronchoalveolar lavage fluid pharmacokinetics of different inhalation doses of PYY(3-36) were compared to IP and subcutaneous efficacious doses. Based on these pharmacokinetic data, inhalation doses of 70:30 PYY(3-36):Dextran T10 were evaluated in a mouse model of appetite suppression and compared to IP and subcutaneous data. RESULTS: Inhalation pharmacokinetic studies showed that plasma exposure was similar for a 2 × higher inhalation dose when compared to subcutaneous and IP delivery. Inhalation doses of 0.22 and 0.65 mg/kg were for efficacy studies. The results showed a dose-dependent (not dose proportional) decrease in food consumption over 4 h, which is similar to IP and subcutaneous delivery routes. CONCLUSIONS: The pharmacokinetic and pharmacodynamics results substantiate the ability of pharmacokinetic data to inform pharmacodynamics dose selection for inhalation delivery of the peptide PYY(3-36). Additionally, engineered PYY(3-36):Dextran T10 particles delivered to the respiratory tract show promise as a non-invasive therapeutic for appetite suppression.

A novel mutation in GJA1 causing oculodentodigital syndrome and primary lymphoedema in a three generation family.

Oculodentodigital syndrome (ODD; OMIM 164200) is a congenital condition with phenotypic features most commonly affecting the face, eyes, dentition and digits. The condition is caused by mutations in the GJA1 gene on chromosome 6. GJA1 codes for connexin 43, a gap junction protein important in providing cell to cell communication and is expressed in lymphatic valves. We present a patient with a clinical and molecular diagnosis of ODD and lower limb lymphoedema. Sanger sequencing of family members confirmed that the missense, p.K206R, GJA1 mutation segregated with the phenotype suggestive of causality. To our knowledge this association has not been reported previously. This is therefore the second connexin gene associated with a lymphoedema phenotype after the recent publication of GJC2 (connexin 47) as a cause of four limb lymphoedema.

The classification and diagnostic algorithm for primary lymphatic dysplasia: an update from 2010 to include molecular findings.

Historically, primary lymphoedema was classified into just three categories depending on the age of onset of swelling; congenital, praecox and tarda. Developments in clinical phenotyping and identification of the genetic cause of some of these conditions have demonstrated that primary lymphoedema is highly heterogenous. In 2010, we introduced a new classification and diagnostic pathway as a clinical and research tool. This algorithm has been used to delineate specific primary lymphoedema phenotypes, facilitating the discovery of new causative genes. This article reviews the latest molecular findings and provides an updated version of the classification and diagnostic pathway based on this new knowledge.

Validating cell-free DNA from supernatant for molecular diagnostics on cytology specimens.

BACKGROUND: Cytology specimens are often used for biomarker testing in the setting of neoplasia. On occasion, formalin-fixed paraffin-embedded (FFPE) cell blocks unfortunately may not yield sufficient material for testing. Recent studies have suggested that residual supernatant fluid from cell block preparation is a valuable source of DNA: both cellular and cell-free DNA (cfDNA). In the present study, the use of cfDNA from supernatant is compared against DNA from FFPE materials. METHODS: cfDNA was extracted prospectively from residual supernatants of 30 cytology samples (29 neoplastic cases and 1 benign ascitic fluid from a patient with a history of melanoma). Samples were tested using clinically validated next-generation-sequencing platforms and the results were compared with data from paired FFPE cell blocks in a real-time prospective clinical setting. Thirteen samples were tested on an amplicon-based assay (Solid Tumor Hotspot), and 17 samples were tested using a comprehensive capture-based assay (UW-Oncoplex). RESULTS: Neoplastic content was estimated by mutational variant allele fraction, with a mean content of 24.0% and 25.8% in supernatant and FFPE, respectively. The variant concordance between paired samples was 90%, and identical results were detected in both supernatant and FFPE samples in 74% of cases. CONCLUSIONS: This study confirmed that cfDNA from supernatant is a viable alternative to FFPE cell blocks for molecular biomarker testing using both amplicon-based and capture-based assays with potential for decreasing additional tissue sampling and faster turnaround time.

A new classification system for primary lymphatic dysplasias based on phenotype.

Traditional classification systems for lymphoedema are of limited use for the diagnosis of specific forms of primary lymphoedema. The understanding of primary lymphoedema has been impeded by confusing terminology and a tendency to simply divide patients into three categories based on the age of onset: lymphoedema congenita manifests at or shortly after birth, lymphoedema praecox is apparent before the age of 35 years and lymphoedema tarda manifests thereafter. The clinical presentation in the spectrum of primary lymphoedema disorders is very variable; the phenotypes of primary lymphoedema conditions vary in the age of onset, site of the oedema, inheritance patterns, associated features and genetic causes. Different inheritance patterns are recognised and there are numerous associated anomalies. Some subgroups, such as Milroy disease and Lymphoedema distichiasis, are well characterised, but others are not. A new clinical classification for primary lymphoedema has been developed as a diagnostic algorithm. Its use is demonstrated on 333 probands referred to our lymphoedema clinic. Grouping patients by accurate phenotyping facilitates molecular investigations, understanding of inheritance patterns, and the natural history of different types of primary lymphoedema. Descriptions of the diagnostic categories, some of which have not been previously clearly defined as distinct clinical entities, are illustrated by clinical cases.

Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations.

OBJECTIVES: The rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented. UW-OncoPlex addresses this through the adoption of a modular panel capable of redesign as significant alterations are identified. We describe the validation of OncoPlex version 6 (OPXv6) for the detection of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), structural variants (SVs), microsatellite instability (MSI), and tumor mutational burden (TMB) in a panel of 340 genes. DESIGN: One hundred twelve samples with diverse diagnoses were comprised of formalin-fixed-paraffin-embedded tissue, fresh-frozen tissue, plasma, peripheral blood, bone marrow, saliva, and cell-line DNA. Libraries were prepared from genomic and cell-free DNA, hybridized to a custom panel of xGen Lockdown probes, and sequenced on Illumina platforms. Sequences were processed through a custom bioinformatics pipeline, and variant calls were compared to prior orthogonal clinical results. RESULTS: Accuracy was 99% for SNVs ≥5% allele frequency, 98% for indels, 97% for SVs, 99% for CNVs, 100% for MSI, and 100% for TMB (compared to previous OncoPlex versions). Library preparation turnaround time decreased by 40%, and sequencing quality improved with a 2.5-fold increase in average sequencing coverage and 4-fold increase in percent on-target. CONCLUSIONS: OPXv6 demonstrates improvements over prior UW-OncoPlex versions including reduced capture cost, improved sequencing quality, and decreased time to results. The modular capture probe design also provides a nimble laboratory response in addressing the expansions necessary to meet the needs of the continuously evolving field of molecular oncology.

High-resolution profiling of human cytomegalovirus cell-free DNA in human plasma highlights its exceptionally fragmented nature.

Human cytomegalovirus (CMV) infections comprise a leading cause of newborn impairments worldwide and are pervasive concerns among the immunocompromised. Quantification of CMV viral loads is increasingly used to guide definitions of CMV disease but standardization of CMV quantitation remains problematic, mostly due to differences in qPCR amplicon sizes between clinical laboratories. Here, we used plasma cfDNA sequencing data from 2,208 samples sent for non-invasive prenatal aneuploidy screening to detect CMV and precisely measure the length of CMV fragments in human plasma. CMV reads were identified in 120 (5.4%) samples. Median cfDNA fragment size derived from CMV was significantly shorter than cfDNA derived from human chromosomes (103 vs 172 bp, p < 0.0001), corresponding to the 3rd percentile of human cfDNA. Sequencing of cfDNA from seven plasma samples from transplant patients positive for CMV confirmed the extraordinarily short nature of CMV cfDNA fragment size with a median length of 149 bp. We further show that these high-resolution measurements of CMV DNA fragment size accurately predict measured discrepancies in serum viral load measurements by different qPCR assays. These results highlight the exceptionally fragmented nature of CMV cfDNA and illustrate the promise of plasma cfDNA sequencing for quantitating viral loads through detection of fragments that would be unrecoverable by qPCR.

Analysis of the coding regions of VEGFR3 and VEGFC in Milroy disease and other primary lymphoedemas.

Milroy disease (hereditary lymphoedema type I, MIM 153100) is a congenital onset primary lymphoedema with autosomal dominant inheritance. Mutations in the gene, vascular endothelial growth factor receptor 3, VEGFR3 (FLT4), are known to cause Milroy disease, but there is uncertainty about the prevalence of VEGFR3 mutations in patients with primary lymphoedema and more specifically in those with a phenotype that resembles Milroy disease. This study aims to address this issue and thereby delineate the Milroy disease phenotype. Fifty-two patients with primary lymphoedema were analysed for mutations in the coding regions of VEGFR3. Patients were divided into four groups: Typical Milroy disease with family history (group I), typical Milroy disease with no family history (group II), atypical Milroy disease (group III), and complex primary lymphoedema (group IV). Results demonstrated that with rigorous phenotyping the likelihood of detecting VEGFR3 mutations is optimised. Mutation prevalence is 75% in typical Milroy patients with a family history (group I) and 68% if positive family history is not a diagnostic criterion. A positive family history is not essential in Milroy disease. The likelihood of detecting VEGFR3 mutations in patients who have a phenotype which is not typical of Milroy disease is very small (<5%). For the 22 mutation positive patients, 14 novel VEGFR3 mutations were identified, two of which were in exon 22 and one in exon 17, confirming that these exons should be included in VEGFR3 analysis. No mutations were found outside the kinase domains, showing that analysis of this part of the gene is not useful for Milroy disease patients. VEGFC, which encodes the ligand for VEGFR3, was sequenced in all patients with typical Milroy disease (groups I and II) and no mutations were identified.

Assignment of two loci for autosomal dominant adolescent idiopathic scoliosis to chromosomes 9q31.2-q34.2 and 17q25.3-qtel.

BACKGROUND: Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity, affecting up to 4% of children worldwide. Familial inheritance of AIS is now recognised and several potential candidate loci have been found. METHODS: We studied 25 multi-generation AIS families of British descent with at least 3 affected members in each family. A genomewide screen was performed using microsatellite markers spanning approximately 10-cM intervals throughout the genome. This analysis revealed linkage to several candidate chromosomal regions throughout the genome. Two-point linkage analysis was performed in all families to evaluate candidate loci. After identification of candidate loci, two-point linkage analysis was performed in the 10 families that segregated, to further refine disease intervals. RESULTS: Significant linkage was obtained in a total of 10 families: 8 families to the telomeric region of chromosome 9q, and 2 families to the telomeric region of 17q. A significant LOD score was detected at marker D9S2157 Z(max) = 3.64 ( theta= 0.0) in a four-generation family (SC32). Saturation mapping of the 9q region in family SC32 defined the critical disease interval to be flanked by markers D9S930 and D9S1818, spanning approximately 21 Mb at 9q31.2-q34.2. In addition, seven other families segregated with this locus on 9q. In two multi-generation families (SC36 and SC23) not segregating with the 9q locus, a maximum combined LOD score of Z(max) = 4.08 ( = 0.0) was obtained for marker AAT095 on 17q. Fine mapping of the 17q candidate region defined the AIS critical region to be distal to marker D17S1806, spanning approximately 3.2 Mb on chromosome 17q25.3-qtel. CONCLUSION: This study reports a common locus for AIS in the British population, mapping to a refined interval on chromosome 9q31.2-q34.2 and defines a novel AIS locus on chromosome 17q25.3-qtel.

Intradural extramedullary metastasis: a review of literature and case report.

Introduction: Intradural extramedullary (IDEM) metastatic disease is infrequently encountered by spine surgeons and consequently poorly understood. Discovery often corresponds with the onset of neurologic symptoms and no consensus exists regarding the importance of complete resection or anticipated postoperative outcome. We aim to elucidate treatment methodologies that exist in the literature. Case presentation: We present a unique case of a 57-year-old male with a known history of esophageal adenocarcinoma, including brain and visceral metastases, who presented with cauda equina syndrome. An IDEM metastatic esophageal adenocarcinoma lesion was identified on advanced imaging and biopsy. This was treated operatively without return of neurologic function. Discussion: We reviewed and summarized the existing literature. Trends are highlighted to further guide surgeons treating this unusual metastatic phenomenon. Conclusion: Intradural metastasis is a harbinger of advanced disease with a poor prognosis regardless of the etiology of the primary lesion. There are a number of proposed mechanisms for metastatic spread with little available literature for surgeon guidance. Most authors are advocates of a palliative, decompressive approach.

Milroy disease and the VEGFR-3 mutation phenotype.

Primary congenital lymphoedema (Milroy disease) is a rare autosomal dominant condition for which a major causative gene defect has recently been determined. Mutations in the vascular endothelial growth factor receptor 3 (VEGFR-3) gene have now been described in 13 families world-wide. This is a review of the condition based on the clinical findings in 71 subjects from 10 families. All 71 individuals have a mutation in VEGFR-3. Ninety per cent of the 71 individuals carrying a VEGFR-3 mutation showed signs of oedema, which was confined in all cases to the lower limbs. In all but two cases onset of swelling was from birth. Other symptoms and signs included cellulitis (20%), large calibre leg veins (23%), papillomatosis (10%), and upslanting toenails (10%). In males, hydrocoele was the next most common finding after oedema (37%). Thorough clinical examination of these patients indicates that there are few clinical signs in addition to lower limb oedema. Rigorous phenotyping of patients produces a high yield of VEGFR-3 mutations.

Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24.

INTRODUCTION: Lymphoedema-distichiasis syndrome (LD) (OMIM 153400) is a rare, primary lymphoedema of pubertal onset, associated with distichiasis. Causative mutations have now been described in FOXC2, a forkhead transcription factor gene. Numerous clinical associations have been reported with this condition, including congenital heart disease, ptosis, varicose veins, cleft palate, and spinal extradural cysts. SUBJECTS: We report clinical findings in 74 affected subjects from 18 families and six isolated cases. All of them were shown to have mutations in FOXC2 with the exception of one family who had two affected subjects with lymphoedema and distichiasis and linkage consistent with the 16q24 locus. RESULTS: The presence of lymphoedema was highly penetrant. Males had an earlier onset of lymphoedema and a significantly increased risk of complications. Lymphatic imaging confirmed the earlier suggestion that LD is associated with a normal or increased number of lymphatic vessels rather than the hypoplasia or aplasia seen in other forms of primary lymphoedema. Distichiasis was 94.2% penetrant, but not always symptomatic. Associated findings included ptosis (31%), congenital heart disease (6.8%), and cleft palate (4%). Other than distichiasis, the most commonly occurring anomaly was varicose veins of early onset (49%). This has not been previously reported and suggests a possible developmental role for FOXC2 in both venous and lymphatic systems. This is the first gene that has been implicated in the aetiology of varicose veins. CONCLUSION: Unlike previous publications, the thorough clinical characterisation of our patients permits more accurate prediction of various phenotypic abnormalities likely to manifest in subjects with FOXC2 mutations.

Detection of six novel FBN1 mutations in British patients affected by Marfan syndrome.

Marfan syndrome (MFS), an autosomal dominant disorder of the extracellular matrix, is due to mutations in fibrillin-1 (FBN1) gene. Investigations carried out in the last decade, unveiled the unpredictability of the site of the mutation, which could be anywhere in the gene. FBN1 mutations have been reported in a spectrum of diseases related to MFS, with no clear evidence for a phenotype-genotype correlation. In this paper we analysed 10 British patients affected by MFS and we were able to characterise five novel missense mutations (C474W, C1402Y, G1987R, C2153Y, G2536R), one novel frameshift mutation (7926delC), one already described mutation (P1424A) and one FBN1 variant (P1148A) classified as a polymorphism in the Asian population. Four out of the five novel missense mutations involved either cysteines or an amino acid conserved in the domain structure. The mutation yield in this study is calculated at 80.0% (8/10), thus indicating that SSCA is a reliable and cost-effective technique for the screening of such a large gene. Our results suggest that this method is reliable to search for FBN1 mutations and that FBN1 screening could be a helpful tool to confirm and possibly anticipate the clinical diagnosis in familial cases. Hum Mutat 18:251, 2001.

Erratum: Detection of six novel FBN1 mutations in British patients affected by Marfan syndrome.

Marfan syndrome (MFS), an autosomal dominant disorder of the extracellular matrix, is due to mutations in fibrillin-1 (FBN1) gene. Investigations carried out in the last decade, unveiled the unpredictability of the site of the mutation, which could be anywhere in the gene. FBN1 mutations have been reported in a spectrum of diseases related to MFS, with no clear evidence for a phenotype-genotype correlation. In this paper we analysed 10 British patients affected by MFS and we were able to characterise five novel missense mutations (C474W, C1402Y, G1987R, C2153Y, G2536R), one novel frameshift mutation (7926delC), one already described mutation (P1424A) and one FBN1 variant (P1148A) classified as a polymorphism in the Asian population. Four out of the five novel missense mutations involved either cysteines or an amino acid conserved in the domain structure. The mutation yield in this study is calculated at 80.0% (8/10), thus indicating that SSCA is a reliable and cost-effective technique for the screening of such a large gene. Our results suggest that this method is reliable to search for FBN1 mutations and that FBN1 screening could be a helpful tool to confirm and possibly anticipate the clinical diagnosis in familial cases.

Expression of the chemokine MIG is a sensitive and predictive marker for antigen-specific, genetically restricted IFN-gamma production and IFN-gamma-secreting cells.

The evaluation of antigen-specific immune responses is critical for understanding the mechanisms of immune protection and for establishing the efficacy of candidate vaccines. Here, we describe a novel assay for IFN-gamma activity which is based on the flow cytometric detection of the chemokine, monokine induced by gamma interferon (MIG) as a sensitive and predictive measure of IFN-gamma-mediated effector function, and a surrogate marker for IFN-gamma-producing cells. Upregulation of MIG expression was demonstrated following in vitro activation of peripheral blood mononuclear cells (PBMCs) with defined CD8+ T-cell epitopes derived from influenza virus, cytomegalovirus (CMV), or Epstein-Barr virus (EBV) and was antigen-specific, genetically restricted and dependent on both CD8+ T cells and IFN-gamma. Furthermore, antigen-specific MIG expression was also demonstrated with Plasmodium falciparum circumsporozoite protein (CSP) peptides, using PBMCs from volunteers immunized with irradiated P. falciparum sporozoites. In multiple parallel experiments, the MIG assay was compared to conventional IFN-gamma ELISPOT, IFN-gamma ELISA, MIG ELISA and intracellular cytokine staining assays. The level of MIG expression was shown to be directly associated with the number of IFN-gamma spot-forming cells (SFCs) detected by ELISPOT (r2=0.94). Moreover, in all instances where cultures were considered positive by ELISPOT, a higher stimulation index was noted with the MIG assay as compared with the ELISPOT assay (on average at least threefold higher) and, in some cases, responses as detected by the MIG assay were significant, but the corresponding response as measured by ELISPOT was not significant. Finally, the flow-based MIG assay offers a number of practical and technical advantages over the ELISPOT assay. Our data validate this novel method for the detection of low as well as high levels of antigen-specific and genetically restricted IFN-gamma activity.

Control mechanisms of virus replication in naturally SIVsmm infected mangabeys and experimentally infected macaques.

As a close cousin to Asian macaques, the African sooty mangabey monkey, a species naturally infected with SIV without ever developing disease, represents an intriguing and thought provoking model for the study of lentiviral infection and disease development. Pursuant to our previous findings that documented the presence of high frequencies of CD8+ T-cells capable of inhibiting lentiviral replication in vitro via a soluble factor, the potential contribution of beta-chemokines and their receptor was evaluated in samples from sooty mangabeys and disease susceptible macaques. Both mangabey and Rhesus macaque PBMC were found to secrete comparable levels of MIP-1alpha, MIP-1beta and RANTES after stimulation in vitro. Constitutive expression of CCR-5 appeared lower in mangabey PBMC but the vast majority of T-cells from mangabeys or macaques were found to express CCR-5 after in vitro activation. Sequence analysis of macaque and mangabey MIP-1alpha and RANTES showed complete homology to the human counterpart. MIP-1beta on the other hand while highly conserved among both monkey species, showed only 93% homology to human MIP-1beta. In addition, CCR-5, CCR-2b and CXCR-4 presented no consistent differences between rhesus and mangabey sequences. Thus, based on current data, differences in disease susceptibility between macaques and mangabeys do not appear to rely on differences in chemokine pathways. However, analyses of the ontogeny of CD8+ T-cell-mediated inhibition of SIV replication showed that macaque PBMC acquire this function shortly after infection, and show a progressive loss thereafter, correlated with progression towards disease. Mangabeys, on the other hand, appear to acquire the CD8+ T-cell inhibitory function long before any evidence of seroconversion to SIV and maintain this function for the lifetime of the animal. In vitro analyses of induction of the CD8+ inhibitory function showed that rhesus CD8+ T-cells have the potential to secrete the inhibitory factor(s) prior to SIV infection.