Vertical stiffness asymmetries during drop jumping are related to ankle stiffness asymmetries.

Asymmetry in vertical stiffness has been associated with increased injury incidence and impaired performance. The determinants of vertical stiffness asymmetry have not been previously investigated. Eighteen healthy men performed three unilateral drop jumps during which vertical stiffness and joint stiffness of the ankle and knee were calculated. Reactive strength index was also determined during the jumps using the ratio of flight time to ground contact time. "Moderate" differences in vertical stiffness (t17  = 5.49; P < 0.001), "small" differences in center of mass displacement (t17  = -2.19; P = 0.043), and "trivial" differences in ankle stiffness (t17  = 2.68; P = 0.016) were observed between stiff and compliant limbs. A model including ankle stiffness and reactive strength index symmetry angles explained 79% of the variance in vertical stiffness asymmetry (R2  = 0.79; P < 0.001). None of the symmetry angles were correlated to jump height or reactive strength index. Results suggest that asymmetries in ankle stiffness may play an important role in modulating vertical stiffness asymmetry in recreationally trained men.

Pulmonary function in an international sample of HIV-positive, treatment-naïve adults with CD4 counts > 500 cells/µL: a substudy of the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial.

OBJECTIVES: The aim of the study was to describe the prevalence and correlates of chronic obstructive pulmonary disease (COPD) in a multicentre international cohort of persons living with HIV (PLWH). METHODS: We performed a cross-sectional analysis of adult PLWH, naïve to HIV treatment, with baseline CD4 cell count > 500 cells/µL enrolled in the Pulmonary Substudy of the Strategic Timing of AntiRetroviral Treatment (START) trial. We collected standardized, quality-controlled spirometry. COPD was defined as forced expiratory volume in 1 s:forced vital capacity (FEV1 :FVC) ratio less than the lower limit of normal. RESULTS: Among 1026 participants from 80 sites and 20 countries, the median age was 36 [interquartile range (IQR) 30, 44] years, 29% were female, and the median time since HIV diagnosis was 1.2 (IQR 0.4, 3.5) years. Baseline median CD4 cell count was 648 (IQR 583, 767) cells/µL, median viral load was 4.2 (IQR 3.5, 4.7) log10 HIV-1 RNA copies/mL, and 10% had a viral load ≤ 400 copies/mL despite lack of HIV treatment. Current/former/never smokers comprised 28%/11%/61% of the cohort, respectively. COPD was present in 6.8% of participants, and varied by age, smoking status and region. Forty-eight per cent of those with COPD reported lifelong nonsmoking. In multivariable regression, age and pack-years of smoking had the strongest associations with FEV1 :FVC ratio (P < 0.0001). There was a significant effect of region on FEV1 :FVC ratio (P = 0.010). CONCLUSIONS: Our data suggest that, among PLWH who were naïve to HIV treatment and had CD4 cell counts > 500 cells/µL, smoking and age were important factors related to COPD. Smoking cessation should remain a high global priority for clinical care and research in PLWH.

Antiviral gene expression in psoriasis.

BACKGROUND: Psoriasis patients have relatively infrequent cutaneous viral infections compared to atopic dermatitis patients. Increased expression of four antiviral proteins (MX1, BST2, ISG15 and OAS2) has been reported in psoriatic skin and genetic studies of psoriasis have identified susceptibility genes in antiviral pathways. OBJECTIVE: To determine if psoriasis is associated with pervasive expression of antiviral genes in skin and blood. METHODS: We performed RNA sequencing on skin samples of 18 subjects with chronic plaque psoriasis and 16 healthy controls. We examined the expression of a predefined set of 42 antiviral genes, each of which has been shown in previous studies to inhibit viral replication. In parallel, we examined antiviral gene expression in atopic dermatitis, non-lesional psoriatic skin and psoriatic blood. We performed HIV-1 infectivity assays in CD4+ peripheral blood T cells from psoriatic and healthy individuals. RESULTS: We observed significant overexpression of 16 antiviral genes in lesional psoriatic skin, with a greater than two-fold increase in ISG15, RSAD2, IRF7, MX2 and TRIM22 (P < 1E-07). None of these genes was overexpressed in atopic dermatitis skin (P < 0.0001) or non-lesional psoriatic skin. In contrast to the skin compartment, no differences in antiviral gene expression were detected in the peripheral blood of psoriasis cases compared to healthy controls. CD4+ T cells from both psoriatic and healthy patients supported HIV-1 infection at a similar rate. CONCLUSION: Our findings highlight psoriasis as an inflammatory disease with cutaneous but not systemic immune activation against viral pathogens.

HIV-1 drug resistance prevalence, drug susceptibility and variant characterization in the Jacobi Medical Center paediatric cohort, Bronx, NY, USA.

OBJECTIVES: With the advent of combined antiretroviral therapy (cART), perinatally HIV-infected children are surviving into adolescence and beyond. However, drug resistance mutations (DRMs) compromise viral control, affecting the long-term effectiveness of ART. The aims of this study were to detect and identify DRMs in a HIV-1 infected paediatric cohort. METHODS: Paired plasma and dried blood spots (DBSs) specimens were obtained from HIV-1 perinatally infected patients attending the Jacobi Medical Center, New York, USA. Clinical, virological and immunological data for these patients were analysed. HIV-1 pol sequences were generated from samples to identify DRMs according to the International AIDS Society (IAS) 2011 list. RESULTS: Forty-seven perinatally infected patients were selected, with a median age of 17.7 years, of whom 97.4% were carrying subtype B. They had a mean viral load of 3143 HIV-1 RNA copies/mL and a mean CD4 count of 486 cells/µL at the time of sampling. Nineteen patients (40.4%) had achieved undetectable viraemia (< 50 copies/mL) and 40.5% had a CD4 count of > 500 cells/µL. Most of the patients (97.9%) had received cART, including protease inhibitor (PI)-based regimens in 59.6% of cases. The DRM prevalence was 54.1, 27.6 and 27.0% for nucleoside reverse transcriptase inhibitors (NRTIs), PIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs), respectively. Almost two-thirds (64.9%) of the patients harboured DRMs to at least one drug class and 5.4% were triple resistant. The mean nucleotide similarity between plasma and DBS sequences was 97.9%. Identical DRM profiles were present in 60% of plasma-DBS paired sequences. A total of 30 DRMs were detected in plasma and 26 in DBSs, with 23 present in both. CONCLUSIONS: Although more perinatally HIV-1-infected children are reaching adulthood as a result of advances in cART, our study cohort presented a high prevalence of resistant viruses, especially viruses resistant to NRTIs. DBS specimens can be used for DRM detection.

NK cells and CD1d-restricted NKT cells respond in different ways with divergent kinetics to IL-2 treatment in primary HIV-1 infection.

Cytokine immunotherapy is being evaluated as adjunct treatment in infectious diseases. The effects on innate and adaptive immunity in vivo are insufficiently known. Here, we investigate whether combination treatment with antiretroviral therapy (ART) and Interleukin-2 (IL-2) of patients with primary HIV-1 infection induces sustained increases in circulating NKT cell and NK cell numbers and effector functions and investigate how changes are coordinated in the two compartments. Patients with primary HIV-1 infection starting ART were analyzed for numbers, phenotype and function of NKT cells, NK cells and dendritic cells (DC) in peripheral blood before, during and after IL-2 treatment. NKT cells expanded during IL-2 treatment as expected from previous studies. However, their response to α-galactosyl ceramide antigen were retained but not boosted. Myeloid DC did not change their numbers or CD1d-expression during treatment. In contrast, the NK cell compartment responded with rapid expansion of the CD56(dim) effector subset and enhanced IFNγ production. Expansions of NKT cells and NK cells retracted back towards baseline values at 12 months after IL-2 treatment ended. In summary, NKT cells and NK cells respond to IL-2 treatment with different kinetics. Effects on cellular function are distinct between the cell types and the effects appear not to be sustained after IL-2 treatment ends. These results improve our understanding of the effects of cytokine immunotherapy on innate cellular immunity in early HIV-1 infection.

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Lower numbers of circulating Natural Killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV-1) associated neurological disease.

Human T lymphotropic virus type 1 (HTLV-1) infects 10-20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV-1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: São Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4(+) and fewer CD8(+) cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4(+) NK T subset are associated with HTLV-1 disease progression.

Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.

Although cytotoxic T lymphocytes (CTLs) are thought to be involved in the control of human immunodeficiency virus-type 1 (HIV-1) infection, it has not been possible to demonstrate a direct relation between CTL activity and plasma RNA viral load. Human leukocyte antigen-peptide tetrameric complexes offer a specific means to directly quantitate circulating CTLs ex vivo. With the use of the tetrameric complexes, a significant inverse correlation was observed between HIV-specific CTL frequency and plasma RNA viral load. In contrast, no significant association was detected between the clearance rate of productively infected cells and frequency of HIV-specific CTLs. These data are consistent with a significant role for HIV-specific CTLs in the control of HIV infection and suggest a considerable cytopathic effect of the virus in vivo.

Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.

HIV-1-specific immune responses in subjects who temporarily contain virus replication after discontinuation of highly active antiretroviral therapy.

Therapeutic intervention with highly active antiretroviral therapy (HAART) can lead to suppression of HIV-1 plasma viremia to undetectable levels for 3 or more years. However, adherence to complex drug regimens can prove problematic, and subjects may temporarily discontinue HAART for variable periods. We studied 6 HIV-1-infected individuals who stopped therapy. Off HAART, levels of viremia were suppressed to fewer than 500 copies/mL in 2 subjects for more than 12 and more than 24 months, respectively, and in 1 subject for 4 months on 1 occasion. Three subjects failed to contain plasma viremia. Broad and strong HIV-1-specific immune responses were detected in subjects with prolonged suppression of viral replication. This longitudinal study suggests that containment of HIV-1 replication to low or undetectable levels after discontinuation of HAART is associated with strong virus-specific immune responses. Boosting of HIV-1-specific immune responses should be considered as an adjunctive treatment strategy for HIV-1-infected individuals on HAART.

Bacteroides are associated with GALT iNKT cell function and reduction of microbial translocation in HIV-1 infection.

Invariant natural killer T (iNKT) cells are innate-like T cells that respond to lipid antigens presented by CD1d. These immunoregulatory cells have the capacity for rapid cytokine release after antigen recognition and are essential for the activation of multiple arms of the immune response. HIV-1 infection is associated with iNKT cell depletion in the peripheral blood; however, their role in the gastrointestinal-associated lymphoid tissue (GALT) is less well studied. Our results show that iNKT cells are found at a higher frequency in GALT compared with blood, particularly in HIV-1 elite controllers. The capacity of iNKT cells to produce interleukin-4 (IL-4) and IL-10 in the GALT was associated with less immune activation and lower markers of microbial translocation, whereas regulatory T cell frequency showed positive associations with immune activation. We hypothesized that the composition of the microbiota would influence iNKT cell frequency and function. We found positive associations between the abundance of several Bacteroides species and iNKT cell frequency and their capacity to produce IL-4 in the GALT but not in the blood. Overall, our results are consistent with the hypothesis that GALT iNKT cells, influenced by certain bacterial species, may have a key role in regulating immune activation in HIV-1 infection.

Hyperalimentation in the undernourished cancer patient.

Serial anthropometrics, creatinine-height ratios. serum albumin levels, and elemental balances were compared for cachectic cancer and noncancer patients receiving hyperalimentation. Cancer patients compared unfavorably in all measurements except triceps skinfold increments, which were equal for both groups, suggesting that weight gain in cancer patients represented repletion of fat rather than restoration of normal lean body mass.

Structures of the oligosaccharide chains of two forms of alpha 1-acid glycoprotein purified from liver metastases of lung, colon, and breast tumors.

Two forms of alpha 1-acid glycoprotein with common immunological determinants and almost identical amino acid compositions but different amounts of carbohydrate were isolated from liver metastases of primary colon, lung, and breast tumors by extraction with perchloric acid, gel filtration on Sepharose CL-6B and Sephadex G-200, and affinity chromatography on concanavalin A:agarose and Ricinus communis agglutinin l:agarose. Both forms of the antigen yielded single bands which stained for protein and carbohydrate when examined by disc gel electrophoresis and immunodiffusion. The molecular weights of the two forms were 45,000 and 37,000 respectively. The larger form contained about five to six oligosaccharide chains, whereas the smaller form had only three to four chains. The composition and structures of the oligosaccharide chains in the two forms of this glycoprotein were very similar. Each contained di-, tri-, and tetraantennary complex-type oligosaccharide chains. The diantennary oligosaccharide chains caused both forms of alpha 1-acid glycoprotein to be retained by concanavalin A-agarose columns. The lower-molecular-weight form contained fewer chains and correspondingly fewer terminal galactosyl residues. This resulted in the separation of this species from the higher-molecular-weight form on columns containing R. communis agglutinin I. Three types of reduced oligosaccharides were released from the light and heavy forms of alpha 1-acid glycoprotein by treatment with alkaline borohydride or by hydrazinolysis. These chains were isolated by chromatography on concanavalin A:agarose and Bio-Gel P-6 columns. The arrangement and linkage of sugars in the purified oligosaccharides were determined by periodate oxidation, sequential hydrolysis with glycosidases, and methylation analysis. The major oligosaccharide chain, comprising 50 to 55% of the carbohydrate, had a triantennary structure as shown in the structure: (formula; see text) in which NeuNAc is N-acetylneuraminic acid, Gal is galactose, GlcNAc is N-acetylglucosamine, Man is mannose, GlcNAcol is N-acetylglucosaminitol, and Fuc is fucose. Tetraantennary chains comprised about 25 to 30% of the carbohydrate, and the additional outer chain was attached to the alpha 1,6-mannosyl residue through a beta 1,6-linked GlcNAc unit. The remaining 15 to 20% of the oligosaccharide chains had a diantennary structure. The extent of sialylation of these chains varied in samples isolated from tumors of the same histological type from different individuals. However, a relatively constant proportion of the three types of chains was present in different forms of the glycoprotein isolated from liver metastases.

Comparison of serine palmitoyltransferase in Morris hepatoma 7777 and rat liver.

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes an initial and regulatory reaction of sphingolipid biosynthesis, the formation of a homologue of 3-ketosphinganine from L-serine and a fatty acyl coenzyme A thioester. We have demonstrated that this enzyme exists in microsomes from Morris hepatoma 7777 and, moreover, found that its specific activity was 199 +/- 23 pmol/min/mg of microsomal protein, which was significantly higher than that for microsomes from host (69 +/- 4.2 pmol/min/mg; S.D.) or control (43 +/- 4.1 pmol/min/mg) rat livers when assayed under optimal conditions. The activities varied with tumor weight. For comparison, the activities of microsomes from regenerating liver were also higher; whereas fetal and neonatal rat livers had substantially lower activities. Assays conducted without added pyridoxal 5'-phosphate revealed that the enzyme in microsomes isolated from the tumor was more readily depleted of this cofactor than was that in control microsomes; this phenomenon was most pronounced with the larger tumors. The other properties of the enzyme resembled those for liver. On the basis of these experiments, we propose that the elevated proportions of sphingomyelin and other sphingolipids found in hepatomas and regenerating rat liver are related to increases in long-chain base synthesis by serine palmitoyltransferase. Since the activity is apparently more sensitive to the availability of pyridoxal 5'-phosphate in the hepatoma, this reaction could become limiting under more severe vitamin B6 deficiencies.

Purification and properties of alpha-D-mannose:beta-1,2-N-acetylglucosaminyl-transferases and alpha-D-mannosidases from human adenocarcinoma.

Two specific N-acetylglucosaminyltransferases, alpha-1,3-mannoside:beta-2-N-acetylglucosaminyltransferase (transferase I) and alpha-1,6-mannoside:beta-2-N-acetylglucosaminyltransferase (transferase II), which catalyze the transfer of N-acetylglucosamine (GlcNAc) from uridine diphospho-GlcNAc to terminal branched alpha-mannosyl (Man) residues, were purified from liver metastases of human colon adenocarcinoma. Transferase I was assayed with Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc beta 1, 4GlcNAc-Asn (Km 0.35 mM), and transferase II was assayed with Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1,4GlcNAc beta 1,4-Glc-NAc-Asn (Km 1.0 mM), in which Asn is asparagine. The Km of transferase I for Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc-beta 1,4)-(Fuc alpha 1,6)GlcNAc-Asn was 1 mM. The specificity of the interaction of transferase I with ovalbumin, ovomucoid, the modified heavy chain of porcine immunoglobulin G and glycopeptides prepared from these glycoproteins was examined by kinetic and structural analysis. The best macromolecular substrates for transferase I were ovalbumin devoid of terminal GlcNAc and some mannose, a solubilized preparation of the heavy chain of porcine immunoglobulin G, devoid of sialic acid, galactose, and terminal GlcNAc, and untreated ovomucoid. The apparent KmS were 45, 19, and 390 microM for ovalbumin, the modified heavy chain of immunoglobulin G, and untreated ovomucoid, respectively. The apparent Km of the enzyme for uridine diphospho-GlcNAc was not significantly influenced by the nature of the glycoprotein acceptor, and it varied between 14 and 20 microM for the different glycoproteins. The structures of the oligosaccharide chains in these glycoproteins which acted as acceptors for the purified enzyme were determined. A major glycopeptide product with the structure Man alpha 1,3(Man alpha 1,6)Man alpha 1,6(14C-GlcNAc beta 1,2Man-alpha 1,3)Man beta 1,4GlcNAc-beta 1,4-GlcNAc-Asn was isolated from both ovalbumin and ovomucoid following incubation with transferase I. The specificity of the enzyme for terminal branched mannosyl residues attached to a beta-linked mannose unit greatly restricts the action of this transferase to this juncture in the synthesis of complex-type oligosaccharide chains of N-asparagine-linked glycoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of autostimulatory and transforming growth factors from human melanoma cells.

Serum-free growth of the human malignant melanoma cell line Hs0294 is associated with production of transforming growth factor-alpha and an autostimulatory melanoma mitogen (melanoma growth-stimulatory activity, MGSA). The transforming activity is characterized by stimulation of anchorage-independent growth of normal rat kidney fibroblasts and competition with 125I-epidermal growth factor for binding to normal rat kidney cells. The second activity, MGSA, stimulates the anchorage-dependent growth of human melanoma cells in serum-free culture medium. When acetic acid extracts of Hs0294 conditioned medium are subjected to Bio-Gel P-30 chromatography followed by reverse-phase high-pressure liquid chromatography, the majority of the transforming growth factor-alpha elutes at 30 +/- 4% acetonitrile, while the major peak of MGSA elutes at 35 +/- 3% acetonitrile. These data indicate that the anchorage-dependent serum-free growth of the Hs0294 human melanoma cell line is apparently dependent upon the autostimulatory melanoma mitogen, MGSA, which is separable from the 125I-epidermal growth factor competing activity produced by these cells.

In vitro growth promotion in human malignant melanoma cells by fibroblast growth factor.

Bovine pituitary fibroblast growth factor (FGF) stimulates the incorporation of [3H]thymidine into DNA in serum-depleted cultures of some but not other human melanoma cells. The melanotic malignant melanoma cell line MIRW exhibited a 40% increase in [3H]thymidine incorporation into DNA and a 48% increase in cell number in response to 3.73 x 10(-9) M FGF. This same concentration of FGF produced a 22% increase in [3H]thymidine incorporation in the melanotic melanoma cell line Hs0294. However, FGF had no effect on the amelanotic melanoma cell line Hs0675, early-passage cultures of a human amelanotic melanoma (W-1), or early-passage cultures of a congenital nevus (N-1).

Extraction of a melanoma growth-stimulatory activity from culture medium conditioned by the Hs0294 human melanoma cell line.

Extracts of conditioned medium (CM) from Hs0294 human malignant melanoma cells stimulate [3H]thymidine incorporation and an increase in cell number in serum-depleted Hs0294 cells. This activity is acid and heat stable, nonproteolytic, protease sensitive, contains disulfide bonds and elutes broadly from a Bio-Gel P-30 column with an approximate molecular weight range of 6,000 to 25,000. Hs0294 CM also stimulates [3H]thymidine incorporation in nonmalignant human nevus cells and normal rat kidney fibroblast cells but not in human fibroblasts. There was only limited competition with 125I-epidermal growth factor in binding assays. Hs0294 CM extracts stimulate anchorage-independent growth in normal rat kidney fibroblast cells in soft agar but not in Hs0294 cells, nevus cells, or human fibroblasts. This second activity elutes from the Bio-Gel P-30 column in two positions with apparent molecular weights of 27,000 and 11,000.

Serum alpha-1 proteinase inhibitor in advanced cancer: mass variants and functionally inert forms.

In 1984, we reported that while immunoreactive levels of serum alpha-1 proteinase inhibitor (API) increased significantly in nine patients with advanced solid tumors, the functional activity of the inhibitor, as measured by the serum trypsin inhibitory capacity, did not increase proportionately. This suggested that a portion of the circulating API was functionally inert. We have now assayed immunoreactive titers and trypsin inhibitory capacity of serum API of 49 patients with advanced carcinomas and 27 healthy controls. Immunoreactive levels of API (expressed as percentage of normal pooled serum which was taken as 100%) in cancer subjects were significantly elevated as compared to normals (mean +/- SE: 233 +/- 9.0% versus 102 +/- 2.0%, P less than 0.05). Although the trypsin inhibitory capacity of the cancer group (16.0 +/- 0.9 units/ml) was significantly elevated (P less than 0.05) as compared to normals (9.9 +/- 0.1 units/ml), this increase was less than that in the immunoreactive titer of API, suggesting the existence of functionally inert API in serum. The fraction of API which was functionally active in this group of cancer patients was 71.0 +/- 3.0% which was significantly less than the normal 98.0 +/- 2.0% (P less than 0.05). In 12 patients followed serially, both immunoreactive levels of API and the trypsin inhibitory capacity increased significantly at the time of clinical progression of disease. There was a significant correlation between increasing absolute granulocyte count and increasing trypsin inhibitory capacity (correlation coefficient 0.66; P less than 0.001). Neither disease progression nor increasing granulocyte count, however, was associated with increasing proportion of functionally inactive API. The inactive form of API had the same molecular weight as the native molecule as shown by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis of cancer sera. Therefore, the inactive form was not due to a complex between API and a tumor-derived protease or to proteolytic fragmentation of the native API. Elastase inhibitory capacity of cancer sera with subactive API was essentially identical with trypsin inhibitory capacity indicating that the active site methionine was not oxidized in the inert API. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis showed that both normal and cancer serum API existed as two mass variants, at Mr 58,000 and 56,000. Both variants formed complexes with elastase and were functionally active.