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Both structurally related forms of transforming growth factor-β (TGF-β types I and II) are potent inhibitors of tumor cell growth in vitro and can also modulate the differentiation of some cells in culture. In this study, we describe the effects of natural and recombinant TGF-βs on the growth and differentiation of a xenograft of human lung adenocarcinoma A549 in male athymic BALB/c mice. Subcutaneous, peritumoral injection of both forms of TGF-β inhibited, in a dose-dependent manner, the growth of established human lung tumors. Histologically, tumors inhibited by TGF-β appeared more differentiated, as judged by reduced mitotic activity and a predominance of highly specialized mucusecreting goblet-like cell types. These findings suggest that TGF-βs can be useful in the development of novel, perhaps less cytotoxic, cancer therapeutic strategies. [J Natl Cancer Inst 81:1182–1185, 1989] [ABSTRACT FROM PUBLISHER]

Abstract Background Genome-wide association studies have identified numerous single nucleotide polymorphisms (SNPs) affecting high density lipoprotein (HDL) or low density lipoprotein (LDL) cholesterol levels; these SNPs may contribute to the genetic basis of vascular diseases. Results We assessed the impact of 34 SNPs at 23 loci on dyslipidemia, key lipid sub-phenotypes, and severe carotid artery disease (CAAD) in a case-control cohort. The effects of these SNPs on HDL and LDL were consistent with those previously reported, and we provide unbiased estimates of the percent variance in HDL (3.9%) and LDL (3.3%) explained by genetic risk scores. We assessed the effects of these SNPs on HDL subfractions, apolipoprotein A-1, LDL buoyancy, apolipoprotein B, and lipoprotein (a) and found that rs646776 predicts apolipoprotein B level while rs2075650 predicts LDL buoyancy. Finally, we tested the role of these SNPs in conferring risk for ultrasonographically documented CAAD stenosis status. We found that two loci, chromosome 1p13.3 near CELSR2 and PSRC1 which contains rs646776, and 19q13.2 near TOMM40 and APOE which contains rs2075650, harbor risk alleles for CAAD. Conclusion Our analysis of 34 SNPs contributing to dyslipidemia at 23 loci suggests that genetic variation in the 1p13.3 region may increase risk of CAAD by increasing LDL particle number, whereas variation in the 19q13.2 region may increase CAAD risk by promoting formation of smaller, denser LDL particles.

Long-read DNA sequencing has recently emerged as a powerful tool for studying both genetic and epigenetic architectures at single-molecule and single-nucleotide resolution. Long-read epigenetic studies encompass both the direct identification of native cytosine methylation and the identification of exogenously placed DNA N 6 -methyladenine (DNA-m6A). However, detecting DNA-m6A modifications using single-molecule sequencing, as well as coprocessing single-molecule genetic and epigenetic architectures, is limited by computational demands and a lack of supporting tools. Here, we introduce fibertools , a state-of-the-art toolkit that features a semisupervised convolutional neural network for fast and accurate identification of m6A-marked bases using Pacific Biosciences (PacBio) single-molecule long-read sequencing, as well as the coprocessing of long-read genetic and epigenetic data produced using either the PacBio or Oxford Nanopore Technologies (ONT) sequencing platforms. We demonstrate accurate DNA-m6A identification (>90% precision and recall) along >20 kb long DNA molecules with an ∼1000-fold improvement in speed. In addition, we demonstrate that fibertools can readily integrate genetic and epigenetic data at single-molecule resolution, including the seamless conversion between molecular and reference coordinate systems, allowing for accurate genetic and epigenetic analyses of long-read data within structurally and somatically variable genomic regions., (© 2024 Jha et al.; Published by Cold Spring Harbor Laboratory Press.)

RNA polymerases must initiate and pause within a complex chromatin environment, surrounded by nucleosomes and other transcriptional machinery. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address this, we employed long-read chromatin fiber sequencing (Fiber-seq) in Drosophila to visualize RNA polymerase (Pol) within its native chromatin context with single-molecule precision along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of individual Pol II, nucleosome, and transcription factor footprints, revealing Pol II pausing-driven destabilization of downstream nucleosomes. Furthermore, we demonstrate pervasive direct distance-dependent transcriptional coupling between nearby Pol II genes, Pol III genes, and transcribed enhancers, modulated by local chromatin architecture. Overall, transcription initiation reshapes surrounding nucleosome architecture and couples nearby transcriptional machinery along individual chromatin fibers., Competing Interests: Declaration of interests A.B.S. is a co-inventor on a patent relating to the Fiber-seq method (US17/995,058)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Since their initial discovery in maize, transposable elements (TEs) have emerged as being integral to the evolution of maize, accounting for 80% of its genome. However, the repetitive nature of TEs has hindered our understanding of their regulatory potential. Here, we demonstrate that long-read chromatin fiber sequencing (Fiber-seq) permits the comprehensive annotation of the regulatory potential of maize TEs. We uncover that only 94 LTR retrotransposons contain the functional epigenetic architecture required for mobilization within maize leaves. This epigenetic architecture degenerates with evolutionary age, resulting in solo TE enhancers being preferentially marked by simultaneous hyper-CpG methylation and chromatin accessibility, an architecture markedly divergent from canonical enhancers. We find that TEs shape maize gene regulation by creating novel promoters within the TE itself as well as through TE-mediated gene amplification. Lastly, we uncover a pervasive epigenetic code directing TEs to specific loci, including that locus that sparked McClintock's discovery of TEs., Competing Interests: Competing interests. A.B.S. is a co-inventor on a patent relating to the Fiber-seq method (US17/995,058).

Accurately quantifying the functional consequences of non-coding mosaic variants requires the pairing of DNA sequence with both accessible and closed chromatin architectures along individual DNA molecules-a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic and epigenomic profiling across targeted >100 kilobase loci with ~10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the DMPK CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated γ-globin ( HBG1 / HBG2 ) promoters in primary human hematopoietic cells induced towards an erythroblast lineage increases the accessibility of the HBG1 promoter as well as neighboring regulatory elements. Overall, we find that these non-protein coding mosaic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant., Competing Interests: Competing interest statement A.B.S. is a co-inventor on a patent relating to the Fiber-seq method (US17/995,058). A.L. is an academic co-founder of Ensoma Inc. The remaining authors declare no competing financial interests.

Thyrotropin (TSH) is the master regulator of thyroid gland growth and function. Resistance to TSH (RTSH) describes conditions with reduced sensitivity to TSH. Dominantly inherited RTSH has been linked to a locus on chromosome 15q, but its genetic basis has remained elusive. Here we show that non-coding mutations in a (TTTG) 4 short tandem repeat (STR) underlie dominantly inherited RTSH in all 82 affected participants from 12 unrelated families. The STR is contained in a primate-specific Alu retrotransposon with thyroid-specific cis-regulatory chromatin features. Fiber-seq and RNA-seq studies revealed that the mutant STR activates a thyroid-specific enhancer cluster, leading to haplotype-specific upregulation of the bicistronic MIR7-2/MIR1179 locus 35 kb downstream and overexpression of its microRNA products in the participants' thyrocytes. An imbalance in signaling pathways targeted by these micro-RNAs provides a working model for this cause of RTSH. This finding broadens our current knowledge of genetic defects altering pituitary-thyroid feedback regulation., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Biallelic pathogenic variants in UQCRFS1 underlie a rare form of isolated mitochondrial complex III deficiency associated with lactic acidosis and a distinctive scalp alopecia previously described in two unrelated probands. Here, we describe a participant in the Undiagnosed Diseases Network (UDN) with a dual diagnosis of two autosomal recessive disorders revealed by genome sequencing: UQCRFS1 -related mitochondrial complex III deficiency and GJA8 -related cataracts. Both pathogenic variants have been reported before: UQCRFS1 (NM_006003.3:c.215-1 G>C, p.Val72_Thr81del10) in a case with mitochondrial complex III deficiency and GJA8 (NM 005267.5:c.736 G>T, p.Glu246*) as a somatic change in aged cornea leading to decreased junctional coupling. A multi-modal approach combining enzyme assays and cellular proteomics analysis provided clear evidence of complex III respiratory chain dysfunction and low abundance of the Rieske iron-sulfur protein, validating the pathogenic effect of the UQCRFS1 variant. This report extends the genotypic and phenotypic spectrum for these two rare disorders and highlights the utility of deep phenotyping and genomics data to achieve diagnosis and insights into rare disease.

Objectives: Transcript sequencing of patient-derived samples has been shown to improve the diagnostic yield for solving cases of suspected Mendelian conditions, yet the added benefit of full-length long-read transcript sequencing is largely unexplored., Methods: We applied short-read and full-length transcript sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis., Results: We identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts the branch point critical for intron 6 splicing. Full-length long-read isoform complementary DNA (cDNA) sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates 5 distinct altered splicing transcripts. All 5 altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A)., Discussion: This case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A., Competing Interests: The authors report no relevant disclosures. Go to Neurology.org/NG for full disclosures., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

SLC1A4 is a trimeric neutral amino acid transporter essential for shuttling L-serine from astrocytes into neurons. Individuals with biallelic variants in SLC1A4 are known to have spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) syndrome, but individuals with heterozygous variants are not thought to have disease. We identify an 8-year-old patient with global developmental delay, spasticity, epilepsy, and microcephaly who has a de novo heterozygous three amino acid duplication in SLC1A4 (L86_M88dup). We demonstrate that L86_M88dup causes a dominant-negative N-glycosylation defect of SLC1A4, which in turn reduces the plasma membrane localization of SLC1A4 and the transport rate of SLC1A4 for L-serine., (© 2023 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Recommendations for laboratories to report incidental findings from genomic tests have stimulated interest in such results. In order to investigate the criteria and processes for assigning the pathogenicity of specific variants and to estimate the frequency of such incidental findings in patients of European and African ancestry, we classified potentially actionable pathogenic single-nucleotide variants (SNVs) in all 4300 European- and 2203 African-ancestry participants sequenced by the NHLBI Exome Sequencing Project (ESP). We considered 112 gene-disease pairs selected by an expert panel as associated with medically actionable genetic disorders that may be undiagnosed in adults. The resulting classifications were compared to classifications from other clinical and research genetic testing laboratories, as well as with in silico pathogenicity scores. Among European-ancestry participants, 30 of 4300 (0.7%) had a pathogenic SNV and six (0.1%) had a disruptive variant that was expected to be pathogenic, whereas 52 (1.2%) had likely pathogenic SNVs. For African-ancestry participants, six of 2203 (0.3%) had a pathogenic SNV and six (0.3%) had an expected pathogenic disruptive variant, whereas 13 (0.6%) had likely pathogenic SNVs. Genomic Evolutionary Rate Profiling mammalian conservation score and the Combined Annotation Dependent Depletion summary score of conservation, substitution, regulation, and other evidence were compared across pathogenicity assignments and appear to have utility in variant classification. This work provides a refined estimate of the burden of adult onset, medically actionable incidental findings expected from exome sequencing, highlights challenges in variant classification, and demonstrates the need for a better curated variant interpretation knowledge base., (© 2015 Amendola et al.; Published by Cold Spring Harbor Laboratory Press.)

Hypertriglyceridemia (HTG) is a heritable risk factor for cardiovascular disease. Investigating the genetics of HTG may identify new drug targets. There are ~35 known single-nucleotide variants (SNVs) that explain only ~10% of variation in triglyceride (TG) level. Because of the genetic heterogeneity of HTG, a family study design is optimal for identification of rare genetic variants with large effect size because the same mutation can be observed in many relatives and cosegregation with TG can be tested. We considered HTG in a five-generation family of European American descent (n = 121), ascertained for familial combined hyperlipidemia. By using Bayesian Markov chain Monte Carlo joint oligogenic linkage and association analysis, we detected linkage to chromosomes 7 and 17. Whole-exome sequence data revealed shared, highly conserved, private missense SNVs in both SLC25A40 on chr7 and PLD2 on chr17. Jointly, these SNVs explained 49% of the genetic variance in TG; however, only the SLC25A40 SNV was significantly associated with TG (p = 0.0001). This SNV, c.374A>G, causes a highly disruptive p.Tyr125Cys substitution just outside the second helical transmembrane region of the SLC25A40 inner mitochondrial membrane transport protein. Whole-gene testing in subjects from the Exome Sequencing Project confirmed the association between TG and SLC25A40 rare, highly conserved, coding variants (p = 0.03). These results suggest a previously undescribed pathway for HTG and illustrate the power of large pedigrees in the search for rare, causal variants., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Blood pressure (BP) is a heritable determinant of risk for cardiovascular disease (CVD). To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP) and pulse pressure (PP), we genotyped ∼50 000 single-nucleotide polymorphisms (SNPs) that capture variation in ∼2100 candidate genes for cardiovascular phenotypes in 61 619 individuals of European ancestry from cohort studies in the USA and Europe. We identified novel associations between rs347591 and SBP (chromosome 3p25.3, in an intron of HRH1) and between rs2169137 and DBP (chromosome1q32.1 in an intron of MDM4) and between rs2014408 and SBP (chromosome 11p15 in an intron of SOX6), previously reported to be associated with MAP. We also confirmed 10 previously known loci associated with SBP, DBP, MAP or PP (ADRB1, ATP2B1, SH2B3/ATXN2, CSK, CYP17A1, FURIN, HFE, LSP1, MTHFR, SOX6) at array-wide significance (P < 2.4 × 10(-6)). We then replicated these associations in an independent set of 65 886 individuals of European ancestry. The findings from expression QTL (eQTL) analysis showed associations of SNPs in the MDM4 region with MDM4 expression. We did not find any evidence of association of the two novel SNPs in MDM4 and HRH1 with sequelae of high BP including coronary artery disease (CAD), left ventricular hypertrophy (LVH) or stroke. In summary, we identified two novel loci associated with BP and confirmed multiple previously reported associations. Our findings extend our understanding of genes involved in BP regulation, some of which may eventually provide new targets for therapeutic intervention.

To identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with ∼2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. In silico follow-up analysis of putative association signals found in independent genome-wide association studies (including 8,130 cases and 38,987 controls) performed by the DIAGRAM consortium identified a T2D locus at genome-wide significance (GATAD2A/CILP2/PBX4; p = 5.7 × 10(-9)) and two loci exceeding study-wide significance (SREBF1, and TH/INS; p < 2.4 × 10(-6)). Second, meta-analyses of 1,986 cases and 7,695 controls from eight African-American studies identified study-wide-significant (p = 2.4 × 10(-7)) variants in HMGA2 and replicated variants in TCF7L2 (p = 5.1 × 10(-15)). Third, conditional analysis revealed multiple known and novel independent signals within five T2D-associated genes in samples of European ancestry and within HMGA2 in African-American samples. Fourth, a multiethnic meta-analysis of all 39 studies identified T2D-associated variants in BCL2 (p = 2.1 × 10(-8)). Finally, a composite genetic score of SNPs from new and established T2D signals was significantly associated with increased risk of diabetes in African-American, Hispanic, and Asian populations. In summary, large-scale meta-analysis involving a dense gene-centric approach has uncovered additional loci and variants that contribute to T2D risk and suggests substantial overlap of T2D association signals across multiple ethnic groups., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Phospholipid transfer protein activity (PLTPa) is associated with insulin levels and has been implicated in atherosclerotic disease in both mice and humans. Variation at the PLTP structural locus on chromosome 20 explains some, but not all, heritable variation in PLTPa. In order to detect quantitative trait loci (QTLs) elsewhere in the genome that affect PLTPa, we performed both oligogenic and single QTL linkage analysis on four large families (n = 227 with phenotype, n = 330 with genotype, n = 462 total), ascertained for familial combined hyperlipidemia. We detected evidence of linkage between PLTPa and chromosome 19p (lod = 3.2) for a single family and chromosome 2q (lod = 2.8) for all families. Inclusion of additional marker and exome sequence data in the analysis refined the linkage signal on chromosome 19 and implicated coding variation in LASS4, a gene regulated by leptin that is involved in ceramide synthesis. Association between PLTPa and LASS4 variation was replicated in the other three families (P = 0.02), adjusting for pedigree structure. To our knowledge, this is the first example for which exome data was used in families to identify a complex QTL that is not the structural locus.

The genetic factors associated with carotid artery disease (CAAD) are not fully known. Because of its role in lipid metabolism, we hypothesized that common genetic variation in the very low density lipoprotein receptor (VLDLR) gene is associated with severe CAAD (>80% stenosis), body mass index (BMI), and lipid traits in humans. VLDLR was resequenced for variation discovery in 92 subjects, and single nucleotide polymorphisms (tagSNPs) were chosen for genotyping in a larger cohort (n = 1,027). Of the 17 tagSNPs genotyped, one tagSNP (SNP 1226; rs1454626) located in the 5' flanking region of VLDLR was associated with CAAD, BMI, and LDL-associated apolipoprotein B (apoB). We also identified receptor-ligand genetic interactions between VLDLR 1226 and APOE genotype for predicting CAAD case status. These findings may further our understanding of VLDLR function, its ligand APOE, and ultimately the pathogenesis of CAAD in the general population.

Inflammatory markers have consistently been associated with vascular disease. Evidence of genetic polymorphisms in inflammatory loci that predict severe carotid artery disease (CAAD) would suggest that this relationship is not secondary to other correlated factors, but related to inflammation itself. We examined the full common genetic variation in 42 inflammatory loci for prediction of severe CAAD versus ultrasound proven controls using a tagSNP approach. For selected loci, monocyte RNA levels were contrasted in subjects with and without CAAD. We confirm the association of IL6(-174), FGB (-455), and ALOX5 with CAAD and show that multiple ALOX5 SNPs independently predict CAAD. We provide evidence for previously unreported associations of SNPs in IL4R, NFKBIA, and PLG with CAAD, and weaker evidence for associations with CSF3, IL10RA, and VCAM1. The NFKBIA and IL10RA expression levels significantly differed between subjects with CAAD and controls. These results support a role for genetic variation related to inflammation in CAAD and a causal role for specific gene products.

Objective: Inflammatory markers are associated with vascular disease; however, variation in the acute phase response (APR) has not been evaluated. We evaluated whether the APR magnitude in men with severe carotid artery disease (CAAD) (>80% stenosis) differed from that of men without stenosis (<15% stenosis)., Methods and Results: White males with (n=43) and without (n=61) severe CAAD receiving clinical influenza vaccinations were recruited. Their baseline and 24-hour after -vaccination blood samples were assayed for C-reactive protein (CRP), IL-6, and serum amyloid-a (SAA). In vivo APR to vaccination was measurable and varied among subjects. Adjusted for age, smoking, oral hypoglycemics, aspirin, and stain use, the relative 24-hour changes in levels of ln(CRP), ln(IL-6), and ln(SAA) were higher in men with CAAD than in men without, but only the SAA response was significant (P=0.02); the relative SAA response was 1.6 (95% confidence interval, 1.1 to 2.5) times higher in men with than without CAAD. The APR for all markers appeared to be independent of baseline levels., Conclusions: Influenza vaccination results in a mild, but measurable, APR in men with and without CAAD. SAA APR variability may be a predictor of severe vascular disease that is independent of basal SAA level.

Paraoxonase 1 (PON1) activity is consistently predictive of vascular disease, although the genotype at four functional PON1 polymorphisms is not. To address this inconsistency, we investigated the role of all common PON1 genetic variability, as measured by tagging single-nucleotide polymorphisms (tagSNPs), in predicting PON1 activity for phenylacetate hydrolysis, LDL susceptibility to oxidation ex vivo, plasma homocysteine (Hcy) levels, and carotid artery disease (CAAD) status. The biological goal was to establish whether additional common genetic variation beyond consideration of the four known functional SNPs improves prediction of these phenotypes. PON2 and PON3 tagSNPs were secondarily evaluated. Expanded analysis of an additional 26 tagSNPs found evidence of previously undescribed common PON1 polymorphisms that affect PON1 activity independently of the four known functional SNPs. PON1 activity was not significantly correlated with LDL oxidative susceptibility, but genotypes at the PON1(-108) promoter polymorphism and several other PON1 SNPs were. Neither PON1 activity nor PON1 genotype was significantly correlated with plasma Hcy levels. This study revealed previously undetected common functional PON1 polymorphisms that explain 4% of PON1 activity and a high rate of recombination in PON1, but the sum of the common PON1 locus variation does not explain the relationship between PON1 activity and CAAD.

Paraoxonase (PON1) is an HDL-associated enzyme. Low PON1 activity predicts vascular disease status and is a more reliable predictor of vascular disease than are functional PON1 genotypes. There is evidence that the relationship of PON1 to vascular disease is, in part, due to its antioxidant activity. However, the physical relationship of PON1 with HDL and the existence of cholesterol pathway regulatory elements at the PON1 locus suggest a further relationship of PON1 with lipoproteins, which may contribute to its role in vascular disease. We investigated the relationship of PON1 activity and genotype to lipid-related traits in 91 Caucasian men with severe carotid artery disease and 184 without vascular disease who were not on lipid-lowering medications. Prior studies of PON1 relationship to lipids have not stratified by disease status.. We found that PON1 activity was correlated with HDL traits in controls and with LDL- and VLDL-related traits in cases. We hypothesize differences in the joint regulation of PON1 and lipoproteins in cases and controls.

Reports of significant reductions in plasma viral load by anti-HIV drugs have raised the possibility that antiviral therapy, if initiated sufficiently early, may result in sustained control of infection and prolonged clinical benefits. We evaluated the effects of intervention coincident with infection using an antiviral nucleoside, d4T, in Macaca nemestrina infected with a highly pathogenic isolate of HIV-2 (HIV-2[287]). Infection with this virus reproducibly results in high viremia and rapid CD4+ cell depletion, allowing a sensitive measurement of the treatment effect on viral load and clinical outcome. Compared to the control group, d4T-treated macaques showed significantly lower (2-3 log10) plasma- and cell-associated viral load. No CD4+ cell decline was observed in the treatment group while on therapy with d4T whereas CD4+ cells of control macaques declined from a preinfection mean of 32% of PBMCs to below 10%. Notably, when d4T treatment was withdrawn after 16 weeks, five of the six macaques continued to control viral load and have maintained normal CD4+ cell level for more than a year. These results demonstrate that early antiviral intervention, even of a limited duration, may constitute an important strategy against lentiviral-induced disease.

By animal-to-animal passage in macaques we derived a pathogenic chimeric simian-human immunodeficiency virus (SHIV) that caused CD4+ T cell loss and AIDS in pigtail macaques and used it to inoculate 20 rhesus and pigtail macaques by the intravaginal and intravenous routes. On the basis of the outcome of infection and patterns of CD4+ T cell loss and viral load, disease was classified into four patterns: acute, subacute, chronic, and nonprogressive infection. During the study period, 15 of the 20 animals developed fatal disease, including AIDS, encephalitis, pneumonia, and severe anemia. Opportunistic pathogens identified in these animals included Pneumocystis, cytomegalovirus, Cryptosporidium, Toxoplasma, and Candida. No single parameter by itself predicted outcome, although a combination of low CD4+ T cell counts in blood, high plasma virus levels, and presence of autoantibodies to red blood cells reliably predicted a fatal outcome. Five animals (25%) died within 3 months of inoculation and constituted the group with acute disease, whereas the nine animals (45%) with subacute disease died between 3 and 8 months postinoculation. This 70% mortality within 8 months is significantly shorter than in HIV-1-infected human beings, of whom 70% develop fatal disease a decade after infection. SHIV infection in macaques provides a useful model with which to evaluate antiviral strategies, combining all the advantages of the SIVmac system, yet using a virus bearing the envelope gene of HIV-1.

Transforming growth factor-beta 1 (TGF-beta 1) has beneficial effects on wound healing. However, the ideal method for its administration to the wound site remains unknown. Our aim was to analyze the release of TGF-beta 1 from different formulations and to study whether the changes in wound healing by TGF-beta 1 depend on its topical delivery system. For the studies the TGF-beta 1 was incorporated into phosphate-buffered saline, into a polyoxamer gel, into DuoDERM hydroactive paste, and into a poly(ethylene oxide) hydrogel. The release of 125I-labeled TGF-beta 1 from carriers was measured in full-thickness wounds in rats and the healing of the wounds was analyzed by histology and wound area measurements. The TGF-beta 1 was released from all formulations at a different rate and in an active form as determined by growth inhibition assay. Wound size measurements and the analysis on the amount of cellular influx, fibroplasia, and granulation tissue showed that a single dose (1 microgram/wound) of locally administered TGF-beta 1 significantly (P < 0.01) enhanced the wound healing. This effect was most prominent with polyoxamer gel formulation, which provided the most sustained release of TGF-beta 1. Our finding that the enhancement in wound healing by TGF-beta 1 was significantly dependent on the carrier used for its topical delivery to the wound site is novel and shows the importance of using adequate delivery systems when growth factors are used to enhance wound repair.

Interleukin-2 (IL-2) is a lymphokine with pleiotropic activities on the immune system. When administered in vivo, besides inducing unrestricted tumor cytotoxicity, it is also responsible for the secondary release of other lymphokines, such as IL-1, TNF, and marrow growth factors, which may mediate some of the clinical toxicities (as well as therapeutic effects) seen during IL-2 immunotherapy. Among the clinical effects of IL-2, we previously reported thrombocytopenia and IL-2-induced in vitro inhibition of platelet aggregation accompanied by rapid secretion of alpha-granule components such as platelet factor 4 (PF4) and beta-thromboglobulin. Platelets constitute one of the largest storage forms of TGF beta. Preliminary evaluation of this factor in patients receiving IL-2 had indicated that plasma TGF beta activity increased in cancer patients following IL-2 therapy. We report a more detailed study of the quantitation of TGF beta activity in the plasma of 23 cancer patients treated with IL-2 immunotherapy. Of interest, we found that although elevation of the bioactive form of TGF beta occurred in most patients during IL-2 therapy, it was significantly higher in patients with clinical regression of tumor (p = .004). In the first 2 weeks of therapy increase of plasma TGF beta activity appeared to correlate with a decrease of platelet counts, suggesting that the factor may derive from the storage form of TGF beta contained therein.

Background/aims: Intestinal mucosa, a tissue in a dynamic state of rapid cellular proliferation, is often adversely affected by cytotoxic drugs. The purpose of this study was to develop an oral delivery system targeting transforming growth factor (TGF) beta 1 locally and analyze its effects on the epithelial stem cells of gastrointestinal mucosa., Methods: Rats were treated with recombinant TGF-beta 1 in alginate beads perorally or with recombinant TGF-beta 1 in phosphate-buffered saline perorally or intraperitoneally. Control animals received phosphate-buffered saline only. The size of the villi was measured. Proliferating and mitotic indices were determined by quantifying immunohistochemical staining for proliferating cell nuclear antigen., Results: Alginate beads released no TGF-beta 1 in acid. However, in pH 7.4, TGF-beta 1 was released in an active form. Histomorphometrical analysis showed a marked reduction in villus height (50%-70%) in the intestinal mucosa of animals treated perorally with recombinant TGF-beta 1 in alginate beads. Also, the proliferating and mitotic indices were significantly reduced (P < 0.01) in these animals as compared with controls and other routes of administration., Conclusions: This study shows that recombinant TGF-beta 1 administered using a novel oral delivery system induces stem cell quiescence in the intestinal mucosa of the rat.

Growth factors have been identified as the primary cause of osteoinduction in bone healing. Transforming growth factor beta (TGF-beta) has been shown to promote bone formation and is present in bone in high quantities. The aims of the present study were to isolate TGF-beta from human bone, demonstrate its biologic activity, and analyze the effects of conventional sterilization techniques on activity. Bone, obtained from femoral heads of five patients (mean age, 70 years) was ground, demineralized, and freeze-dried, and samples from each patient were divided into three groups: no treatment, sterilization with 1.60 to 1.94 Mrad of 60Co irradiation, and sterilization with ethylene oxide (ETO). Carrier-free recombinant TGF-beta control was also treated and was totally inactivated by ETO but not by irradiation (p < 0.01). TGF-beta activity in demineralized bone was not significantly diminished (p > 0.1) by either sterilization procedure, and substantial amounts of active TGF-beta were recovered in all bone samples: 1.04 +/- 0.77 ng per mg of protein in irradiated samples, 0.67 +/- 0.26 ng per mg in ETO-treated samples, and 1.04 +/- 0.33 in untreated samples, respectively (mean +/- SD). Although a recent report demonstrated that the osteoinductive activity of bone morphogenetic protein in bone powder is diminished considerably by ETO and by 2.5 Mrad of irradiation sterilization of bone powder, these data demonstrate that TGF-beta activity, with its osteoinductive properties, was not destroyed in more coarsely ground, demineralized bone by ETO or by lower doses of irradiation. These findings support the use of human bone allografts in clinical instances involving impaired bone formation.

Although bone has a remarkable capacity for regenerative growth, there are many clinical situations in which the bony repair process is impaired. TGF-beta 1 is a 25 kD homodimeric protein which modulates the growth and differentiation of many cell types. The ability of TGF-beta 1 to promote bone formation suggests that it may have potential as a therapeutic agent in disease of bone loss. However, there still exists a need for an effective method of delivering TGF-beta 1 to the site of an osseous defect for the promotion of bone healing. This paper describes a novel biodegradable controlled release system for TGF-beta 1 comprised of poly (DL-lactic-co-glycolic acid) (PLPG) and demineralized bone matrix (DBM). The amount and activity of TGF-beta 1 released was determined using several methods including 125I-labeled TGF-beta 1 as a tracer, an enzyme linked immunosorbent assay (ELISA) and a growth inhibitory assay (GIA). Protein was released from the devices for time periods of more than 600 h. The amount of TGF-beta 1 released was directly proportional to both the TGF-beta 1 loading and the weight percent of DBM in the device. The release kinetics could be further controlled by applying polymeric coatings of varying porosity to the devices. The GIA indicated that between 80 and 90% of the TGF-beta 1 released from the delivery system retained its bioactivity. The PLPG and DBM existed in phase separated domains within the device as determined by differential scanning calorimetry. Scanning electron microscopy suggested that the devices were sufficiently porous to allow bone ingrowth.

Transforming growth factor (TGF) activities could be detected in the urine of normal, pregnant, and tumor-bearing humans. These acid- and heat-stable polypeptides competed for binding to epidermal growth factor (EGF) membrane receptors and promoted the anchorage-independent growth of nontransformed rodent cells. They differed from human EGF in their apparent molecular weights and soft-agar growth-stimulating activity. The urine from pregnant females contained TGF activities with apparent molecular weight(s) (relative) (Mr) of 10,000 ad 17,000--20,000. In the case of a lung cancer patient, an additional major activity of approximately 30,000--35,000 Mr was found. All urine specimens examined also contained a "common" 8,000-Mr soft-agar growth-stimulating activity, which competed for binding to EGF membrane receptors and which was chromatographically separable from EGF (urogastrone). Thus urine may provide a convenient and readily available source for the biochemical characterization of these TGF-like activities, some of which may be clinically useful biologic markers for certain types of cancer.