AA amyloidosis associated with morbid obesity (clinical case) / Amiloidosis AA asociada a la obesidad mórbida (caso clínico)

We present the case of a 45-year-old woman who was hospitalized due to severe macrocytic anemia and renal failure. The patient presented a morbid obesity. The immunological study showed anti-ENA anti-SSA (Ro52) positive, with negative antinuclear antibodies. Also in the proteinogram (serum immunofixation) the presence of monoclonal bands IgG lambda and IgG kappa, monoclonal component 7.2% (4.68g/L), with elevation of free light chains (kappa 95.94mg/L (3.3-19.4), evidenced, lambda 145.17mg/L (5.71-26.3)). The bone marrow study showed an infiltration of 5% of plasma cells and positive for AA amyloid. Finally, a percutaneous renal biopsy was performed, which again showed amyloid infiltration. In the genetic study, 2 mutations of the family Mediterranean fever gene (MEFV) have been identified. Secondary AA amyloidosis has been described associated with obesity, in addition to a percentage of cases of unknown etiology

Presentamos el caso de una mujer de 45 años que fue hospitalizada debido a una anemia macrocítica severa e insuficiencia renal. El paciente presentaba una obesidad mórbida. El estudio inmunológico mostró positividad para anti-ENA, anti-SSA (Ro52) y negatividad para anticuerpos antinucleares. También en el proteinograma (inmunofijación sérica) se detectó la presencia de bandas monoclonales IgG lambda e IgG kappa, con un componente monoclonal del 7,2% (4,68g/l) y la elevación de cadenas ligeras libres (kappa 95,94mg/l [3,3-19,4]; lambda 145,17mg/l [5,71-26,3]). El estudio de biopsia de médula ósea mostró una infiltración del 5% de células plasmáticas y positividad para amiloide AA. Finalmente, se realizó una biopsia renal que nuevamente mostró infiltración amiloide. En el estudio genético se identificaron 2 mutaciones del gen de la fiebre mediterránea familiar (MEFV). La amiloidosis secundaria AA se ha descrito asociada a la obesidad, además de un porcentaje de casos de etiología desconocida

Relationship between gut microbiota and circulating metabolites in population-based cohorts.

Gut microbiota has been implicated in major diseases affecting the human population and has also been linked to triglycerides and high-density lipoprotein levels in the circulation. Recent development in metabolomics allows classifying the lipoprotein particles into more details. Here, we examine the impact of gut microbiota on circulating metabolites measured by Nuclear Magnetic Resonance technology in 2309 individuals from the Rotterdam Study and the LifeLines-DEEP cohort. We assess the relationship between gut microbiota and metabolites by linear regression analysis while adjusting for age, sex, body-mass index, technical covariates, medication use, and multiple testing. We report an association of 32 microbial families and genera with very-low-density and high-density subfractions, serum lipid measures, glycolysis-related metabolites, ketone bodies, amino acids, and acute-phase reaction markers. These observations provide insights into the role of microbiota in host metabolism and support the potential of gut microbiota as a target for therapeutic and preventive interventions.

One-step production of bioactive human lipopolysaccharide binding protein from LPS-eliminated E. coli.

Human lipopolysaccharide (LPS) binding protein (LBP) is a ∼60 kDa glycosylated protein that mediates potent innate immune against invading Gram-negative bacteria by recognition of LPS in their outer membranes. To date, there is no method for efficient production of bioactive LPS-free LBP at sufficient amounts through prokaryotic expression system. Here we present a simple approach for rapid preparation of human LBP from a LPS-eliminated E. coli strain named ClearColi BL21 (DE)3. Combined with the usage of an ultra-high-affinity CL7/Im7 purification system, we achieved one-step purification of recombinant human LBP with over 90% purity at a yield of ∼4 mg/L when using LB culture medium. The produced LBP retains full LPS binding activity which was validated by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Collectively, we develop a valid method that can be applied to cost-effectively produce and purify LPS-free proteins.

Evaluación analítica del inmunoanálisis Lumipulse(R) G BRAHMS PCT para la medida de procalcitonina / Analytical evaluation of the Lumipulse G BRAHMS PCT procalcitonin immunoassay

Introducción: La procalcitonina (PCT) es un biomarcador útil para el manejo de pacientes con infección bacteriana severa y sepsis. Actualmente, diversas metodologías están disponibles para su medición. El objetivo de este estudio fue evaluar el rendimiento analítico del nuevo inmunoanálisis Lumipulse(R) BRAHMS PCT, adaptado al analizador Lumipulse G 600II de Fujirebio. Material y métodos: La evaluación analítica incluyó el cálculo del límite de blanco, límite de detección, sensibilidad funcional, imprecisión intraserial y en el laboratorio, la verificación de la linealidad y la comparación con el ensayo ELECSYS(R) BRAHMS PCT. Resultados: El límite de blanco, el límite de detección y la sensibilidad funcional fueron 0,0011 ng/mL, 0,0025 ng/mL y 0,008 ng/mL, respectivamente. La imprecisión intraserial y la imprecisión en el laboratorio variaron entre 0,78 y 2,16 y entre 1,31 y 2,06, respectivamente, utilizando los materiales de control comerciales. La linealidad fue excelente (r=0,999) en el rango de concentraciones establecido por el fabricante. En el análisis de comparación entre métodos, los resultados fueron transferibles entre ambos (Lumipulse(R) BRAHMS PCT = −0,016 + 1,006 * ELECSYS(R) BRAHMS PCT). La diferencia media entre ambos métodos fue 0,2 ng/mL (IC95%: −0,906 a 0,430). Cuando las concentraciones de PCT fueron estratificadas según los rangos de concentraciones habitualmente utilizados para su interpretación clínica, el grado de concordancia fue muy alto (índice kappa: 0,9874 (IC95%: 0,9696 a 1,0000). Conclusión: El nuevo ensayo Lumipulse(R) BRAHMS PCT, con tecnología de quimioluminiscencia enzimática (CLEIA), es aceptable para su uso clínico

Introduction: Procalcitonin (PCT) is a useful biomarker for the management of patients with severe bacterial infection and sepsis. Different types of assays are currently available for its measurement. This study presents an evaluation of the analytical performance of the novel Lumipulse G BRAHMS PCT(TM) immunoassay on the Lumipulse 600II analyser. Material and methods: This analytical evaluation included the calculation of the limit of blank, limit of detection, functional sensitivity, intra-assay and total imprecision, confirmation of linearity and the comparison with the ELECSYS BRAHMS PCT(TM) assay. Results: Limit of blank, limit of detection and functional sensitivity were 0.0011 ng/mL, 0.0025 ng/mL, and 0.008 ng/mL, respectively. Intra-assay and total imprecision ranged from 0.78 to 2.16 and from 1.31 to 2.06, respectively, when control levels were used. The linearity was excellent (r=0.999) in the range of concentrations established by manufacturer. A highly significant agreement was found in the comparison between both assays (Lumipulse BRAHMS PCT = −0.016 + 1.006 * ELECSYS BRAHMS PCT). The mean bias was 0.2 ng/mL (95% CI: −0.906 to 0.430). When PCT levels were stratified according to the ranges normally used for their clinical interpretation, the agreement was very high (kappa index: 0.9874 (95% CI: 0.9696 to 1.0000). Conclusion: The novel assay Lumipulse BRAHMS PCT, with CLEIA technology, appears to be acceptable for clinical use

LpA-II:B:C:D:E: a new immunochemically-defined acute phase lipoprotein in humans.

BACKGROUND: Previous studies of lipoproteins in patients with sepsis have been performed on density fractions isolated by conventional ultracentrifugation that are heterogeneous and provide no information about the cargo of apoproteins present in the immunochemically distinct subclasses that populate the density classes. Since apoproteins are now known to have important roles in host defense, we have separated these subclasses according to their apoprotein content and characterized their changes during experimental endotoxemia in human volunteers. METHODS: We have studied apoB- and apoA containing lipoprotein subclasses in twelve healthy male volunteers before and for 8 h after a single dose of endotoxin (ET; 2 µg/kg) to stimulate inflammation. RESULTS: After endotoxin, TG, TC, apoB and the apoB-containing lipoprotein cholesterol-rich subclass LpB and two of the three triglyceride-rich subclasses (TGRLP: Lp:B:C, LpB:C:E+ LpB:E) all declined. In contrast, the third TGRLP, LpA-II:B:C:D:E ("complex particle"), after reaching a nadir at 4 h rose 49% above baseline, p = .006 at 8 h and became the dominant particle in the TGRLP pool. This increment exceeds the threshold of > 25% change required for designation as an acute phase protein. Simultaneous decreases in LpA-I:A-II and LpB:C:E + LpB:E suggest that these subclasses undergo post-translational modification and contribute to the formation of new LpA-II:B:C:D:E particles. CONCLUSIONS: We have identified a new acute phase lipoprotein whose apoprotein constituents have metabolic and immunoregulatory properties applicable to host defense that make it well constituted to engage in the APR.

Identification of Host Defense-Related Proteins Using Label-Free Quantitative Proteomic Analysis of Milk Whey from Cows with Staphylococcus aureus Subclinical Mastitis.

Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516.

High-efficiency secretory expression of human neutrophil gelatinase-associated lipocalin from mammalian cell lines with human serum albumin signal peptide.

Human neutrophil gelatinase associated lipocalin (NGAL) is a secretory glycoprotein initially isolated from neutrophils. It is thought to be involved in the incidence and development of immunological diseases and cancers. Urinary and serum levels of NGAL have been investigated as a new biomarker of acute kidney injury (AKI), for an earlier and more accurate detection method than with creatinine level. However, expressing high-quality recombinant NGAL is difficult both in Escherichia coli and mammalian cells for the low yield. Here, we cloned and fused NGAL to the C-terminus of signal peptides of human NGAL, human interleukin-2 (IL2), gaussia luciferase (Gluc), human serum albumin preproprotein (HSA) or an hidden Markov model-generated signal sequence (HMM38) respectively for transient expression in Expi293F suspension cells to screen for their ability to improve the secretory expression of recombinant NGAL. The best results were obtained with signal peptide derived from HSA. The secretory recombinant protein could react specifically with NGAL antibody. For scaled production, we used HSA signal peptide to establish stable Chinese hamster ovary cell lines. Then we developed a convenient colony-selection system to select high-expression, stable cell lines. Moreover, we purified the NGAL with Ni-Sepharose column. The recombinant human NGAL displayed full biological activity. We provide a method to enhance the secretory expression of recombinant human NGAL by using the HSA signal peptide and produce the glycoprotein in mammalian cells.

[Prokaryotic expression and immunological activity of human neutrophil gelatinase associated lipocalin].

OBJECTIVE: To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein. METHODS: The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry). RESULTS: Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically. CONCLUSION: Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.

Ovocalyxin-36 is a pattern recognition protein in chicken eggshell membranes.

The avian eggshell membranes are essential elements in the fabrication of the calcified shell as a defense against bacterial penetration. Ovocalyxin-36 (OCX-36) is an abundant avian eggshell membrane protein, which shares protein sequence homology to bactericidal permeability-increasing protein (BPI), lipopolysaccharide-binding protein (LBP) and palate, lung and nasal epithelium clone (PLUNC) proteins. We have developed an efficient method to extract OCX-36 from chicken eggshell membranes for purification with cation and anion exchange chromatographies. Purified OCX-36 protein exhibited lipopolysaccharide (LPS) binding activity and bound lipopolysaccharide (LPS) from Escherichia coli O111:B4 in a dose-dependent manner. OCX-36 showed inhibitory activity against growth of Staphylococcus aureus ATCC 6538. OCX-36 single nucleotide polymorphisms (SNPs) were verified at cDNA 211 position and the corresponding proteins proline-71 (Pro-71) or serine-71 (Ser-71) were purified from eggs collected from genotyped hens. A significant difference between Pro-71 and Ser-71 OCX-36 for S. aureus lipoteichoic acid (LTA) binding activity was detected. The current study is a starting point to understand the innate immune role that OCX-36 may play in protection against bacterial invasion of both embryonated eggs (relevant to avian reproductive success) and unfertilized table eggs (relevant to food safety).

Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C-reactive protein, for clinical use.

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL-6 or IL-8, nor does SAP cause release of IL-1ß or IL-10. Furthermore neither of our preparations was pro-inflammatory in mice in vivo.

Human biomarker discovery and predictive models for disease progression for idiopathic pneumonia syndrome following allogeneic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (SCT) is the only curative therapy for many malignant and nonmalignant conditions. Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication that limits successful outcomes. Preclinical models suggest that IPS represents an immune mediated attack on the lung involving elements of both the adaptive and the innate immune system. However, the etiology of IPS in humans is less well understood. To explore the disease pathway and uncover potential biomarkers of disease, we performed two separate label-free, proteomics experiments defining the plasma protein profiles of allogeneic SCT patients with IPS. Samples obtained from SCT recipients without complications served as controls. The initial discovery study, intended to explore the disease pathway in humans, identified a set of 81 IPS-associated proteins. These data revealed similarities between the known IPS pathways in mice and the condition in humans, in particular in the acute phase response. In addition, pattern recognition pathways were judged to be significant as a function of development of IPS, and from this pathway we chose the lipopolysaccaharide-binding protein (LBP) protein as a candidate molecular diagnostic for IPS, and verified its increase as a function of disease using an ELISA assay. In a separately designed study, we identified protein-based classifiers that could predict, at day 0 of SCT, patients who: 1) progress to IPS and 2) respond to cytokine neutralization therapy. Using cross-validation strategies, we built highly predictive classifier models of both disease progression and therapeutic response. In sum, data generated in this report confirm previous clinical and experimental findings, provide new insights into the pathophysiology of IPS, identify potential molecular classifiers of the condition, and uncover a set of markers potentially of interest for patient stratification as a basis for individualized therapy.

Removal of neutrophil gelatinase-associated lipocalin by extracorporeal therapies.

Neutrophil gelatinase-associated lipocalin (NGAL) protein is an early biomarker for acute kidney injury (AKI). It is unknown if extracorporeal therapies (EC) have an effect on circulating NGAL levels. This study was designed to describe the kinetics of NGAL molecule in different EC techniques and to evaluate NGAL clearance in different operational conditions. A mock hemofiltration (HF) and hemoperfusion (HP) setup was used. NGAL was added to the blood reservoir and then measured at 30-minute intervals from arterial, venous, and ultrafiltrate (UF) lines. Removal kinetics and NGAL sieving coefficient were calculated. In our experiments, baseline NGAL concentration averaged 452 microg/L. There was a consistent downward trend throughout the experiment. NGAL concentration in the UF was between 80 and 90 microg/L, though it showed a slight increase in the second hour. The sieving coefficient of NGAL ranged from 0.2 to 0.4 during HF and it appeared to increase with time, suggesting an initial effect of membrane adsorption. HP proved clearly that there was adsorption of NGAL by the membrane and the point of saturation occured at approximately 60 minutes from the start of circulation. Our evaluation demonstrates that NGAL can be adsorbed and ultrafiltrated with polysulfone membranes. This should be taken into consideration when using NGAL as an AKI biomarker in patients undergoing EC circulation.

Up-regulation of neutrophil gelatinase-associated lipocalin in cholesteatoma.

CONCLUSION: Neutrophil gelatinase-associated lipocalin (NGAL) and Ki-67 expression were up-regulated in cholesteatoma and the expression pattern of NGAL in the epithelial layer was inversely related to the expression of Ki-67. Therefore, NGAL may be related to dysregulated differentiation in the keratinocytes during the development of a cholesteatoma. OBJECTIVES: We investigated the differential expression and localization of NGAL in middle ear cholesteatoma and compared the results to normal external auditory canal (EAC) skin. We also compared the expression and localization of NGAL with the expression and localization of Ki-67 in middle ear cholesteatoma. SUBJECTS AND METHODS: Tissue samples from middle ear cholesteatomas and normal EAC skin were obtained from 20 patients undergoing middle ear surgery. NGAL mRNA expression was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of NGAL protein was analyzed by Western blot. NGAL and Ki-67 were localized by immunohistochemical staining. RESULTS: A significantly greater expression of the NGAL mRNA was observed in cholesteatoma epithelium than in normal EAC skin (p < 0.05). NGAL was detected in the granular layer of cholesteatoma. However, NGAL was scarcely expressed in normal EAC skin. Ki-67 was detected predominantly in the basal and parabasal layers of cholesteatoma epithelium.

Heparin cryoprecipitation reduces plasma levels of non-traditional risk factors for atherosclerosis in vitro.

AIMS: To show that heparin cryoprecipitation (HCP), an in vitro method of plasma purification, reduces the levels of in vivo modified proteins and non-traditional risk factors from plasma of atherosclerotic hemodialysis (HD) patients. METHODS: HCP was applied to plasma obtained from HD patients and controls, forming a precipitate--cryogel. Levels of fibrinogen, albumin, CRP, TNF-alpha, IL-6, advanced oxidation protein products, carbonylated fibrinogen and carbonylated albumin were determined in plasma before and after applying HCP and in the cryogel. RESULTS: Treatment of HD plasma with HCP, beyond the significant reduction of the increased levels of all the above-mentioned molecules, reduced fibrinogen, TNF-alpha, carbonylated fibrinogen and carbonylated albumin to control levels which were simultaneously found in the cryogel. CONCLUSIONS: HCP applied to plasma enables the simultaneous precipitation of modified molecules and circulating non-traditional risk factors for atherosclerosis. This study may serve as a base for the future development of a clinical purification technique.

Immunohistochemical localization of haptoglobin in porcine lungs.

The extravasation of erythrocytes into the lower respiratory tract occurs in numerous lung injuries and may lead to oxidative damages in lung tissues. Haptoglobin (Hp), the major haemoglobin-binding protein, is known to reduce lung injury associated with exposure to blood in mice. In pigs, Hp is a major acute phase protein and its serum concentrations are elevated in various infections of the respiratory tract. However, information on the porcine Hp response towards inflammatory stimuli is restricted to blood. We herein investigated the presence of Hp in lung tissues from pigs with acute and chronic bronchopneumonia via immunohistochemistry. Hp was localized in airway epithelial cells and immigrated leucocytes whereas in alveolar epithelial cells there was no distinct signal. Unaltered lungs showed less Hp-positive cells compared with lungs from pigs with acute or chronic bronchopneumonia.

Acute-phase protein alpha-1-acid glycoprotein mediates neutrophil migration failure in sepsis by a nitric oxide-dependent mechanism.

The reduction of circulating neutrophil migration to infection sites is associated with a poor outcome of severe sepsis. alpha-1-Acid glycoprotein (AGP) was isolated from the sera of severely septic patients by HPLC and acrylamide gel electrophoresis and identified by mass spectrometry. Both the isolated protein and commercial AGP inhibited carrageenin-induced neutrophil migration into the rat peritoneal cavity when administered i.v. at a dose of 4.0 microg per rat (95 pmol per rat). Analysis by intravital microscopy demonstrated that both proteins inhibited the rolling and adhesion of leukocytes in the mesenteric microcirculation. The inhibitory activity was blocked by 50 mg/kg aminoguanidine, s.c., and was not demonstrable in inducible nitric oxide synthase (iNOS) knockout mice. Incubation of AGP with neutrophils from healthy subjects induced the production of NO and inhibited the neutrophil chemotaxis by an iNOS/NO/cyclic guanosine 3,5-monophosphate-dependent pathway. In addition, AGP induced the l-selectin shedding by neutrophils. The administration of AGP to rats with mild cecal ligation puncture sepsis inhibited neutrophil migration and reduced 7-day survival from approximately 80% to 20%. These data demonstrate that AGP, an acute-phase protein, inhibits neutrophil migration by an NO-dependent process and suggest that AGP also participates in human sepsis.

Co-expression by keratinocytes of migration stimulating factor (MSF) and a functional inhibitor of its bioactivity (MSFI).

Migration stimulating factor (MSF) is a potent autocrine and paracrine factor expressed by fibroblasts and epithelial cells in foetal skin, tumours and healing wounds. In tissue culture, MSF bioactivity is present in the conditioned medium of foetal and tumour derived fibroblasts, but not in normal adult fibroblasts or keratinocytes. The conditioned medium of early passage keratinocytes or a keratinocyte line (HaCaT) effectively inhibited the motogenic activity of rhMSF. Fractionation of keratinocyte conditioned medium by size-exclusion chromatography revealed the presence of bioactive MSF as well as a functional inhibitor of MSF (MSFI) in fractions corresponding to approximately 70 kDa and 25 kDa, respectively. MSFI was purified and identified as neutrophil gelatinase-associated lipocalin (NGAL or lipocalin-2). Immunostaining confirmed that keratinocytes expressed both MSF and NGAL, whereas normal adult fibroblasts did not express either. Recombinant and cell-produced NGAL neutralised the motogenic activity of rhMSF. NGAL is known to bind MMP-9 and promote the activity of this protease. In contrast, there was no evidence of NGAL-MSF binding in keratinocyte conditioned medium. MSF displays a number of bioactivities of relevance to cancer progression and wound healing. Our findings indicate a novel function of NGAL and a possible mechanism for regulating MSF activity in tissues.

Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series.

Monitoring changes in serum protein expression in response to acute events such as trauma, infection or drug intervention may reveal key proteins of great value in predicting recovery or treatment response. Concerted actions of many proteins are expected. Proteins sharing similar expression changes may function in the same physiological process. As a model we analyzed expression changes in serum of colon cancer patients, before, during, and after laparoscopic colon resection. Eight samples were taken from each of four patients before, during, and up to 5 days after surgery. Total serum and a low molecular weight fraction were analyzed by SELDI-TOF-MS. In total 146 masses were detected. A principal components analysis (PCA) illustrates the temporal variation in the postsurgery proteome. Time series for each mass could be clustered into four distinct groups based on similarity in expression pattern. Two masses of 11.4 and 11.6 kDa, part of a slow response cluster, were identified as forms of the acute phase protein serum amyloid A (SAA). Fourteen more proteins belong to this cluster and may also function in acute phase response. We present an approach to analyze temporal variation in the proteome. This approach may be useful to evaluate surgical, nutritional, and pharmacological interventions.

Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells.

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.

Identification of lipopolysaccharide-binding proteins in porcine milk.

Septicemia and endotoxemia initiated by bacterial lipopolysaccharide (LPS) are relatively common in suckling and weaned piglets. Maternal milk is a source of both nutrition and immune protection for piglets. Passive transfer of colostral antibodies is necessary for protection of neonatal piglets against diseases, but the concentration of immunoglobulins in milk rapidly declines during the 1st wk of lactation in all mammals. We hypothesized, therefore, that nonimmunoglobulin substances in milk contribute to the innate protection of neonates against septicemia during the suckling period. Using LPS-affinity chromatography for isolation of LPS-binding proteins and liquid chromatography-mass spectrometry for their identification, we identified in porcine milk the following proteins with LPS-binding capacity: lactoferrin, soluble CD14, serum amyloid A, alpha-S1 casein, beta-casein, and kappa-casein. For lactoferrin, alpha-S1 casein, and kappa-casein, in vitro pepsin digestion did not inhibit LPS-binding activity, whereas combined digestion with pepsin and pancreatin abolished it. The biologic functions of these LPS-binding proteins and peptides were not determined.