Prevalence of malnutrition and its associated outcomes in pediatric patients with scoliosis undergoing elective posterior spinal fusion or spine growth modulation - a retrospective review.

Recent attention within pediatric orthopedics focuses on the prevalence and prevention of post-operative complications, including surgical site infections (SSIs). While poor nutrition status has been noted as a risk factor, various definitions have been utilized. The aim of this retrospective chart review was to utilize the Academy of Nutrition and Dietetics (AND) and the American Society for Parenteral and Enteral Nutrition (ASPEN) diagnostic criteria to determine both the prevalence of malnutrition in pediatric patients undergoing spine deformity surgery and its influence on the prevalence of post-operative complications. A total of 2603 patients had a spine procedure between 2012 and 2018. Patients were excluded if they were less than 2 years of age or greater than 18 years of age and/or did not have their spine procedure completed at Children's Wisconsin. Patients who met inclusion criteria and had an irrigation and debridement (I&D) were selected for an I&D group. From the remaining charts, 127 patients were randomly selected for the non-I&D group. Patients in both groups were further divided into well-nourished and malnourished groups. T-tests and chi square tests were used to determine statistical significance. We found that 50% of patients who had an I&D had malnutrition during their clinical course. This is compared with 17% of patients who didn't require an I&D. Additionally, patients requiring multiple surgical interventions, had an increased prevalence of malnutrition. With the recent focus on reducing the prevalence of post-operative complications, the identification and treatment of malnutrition may be helpful in reducing post-operative complications.

A modified caprylic acid method for manufacturing immunoglobulin G from human plasma with high yield and efficient virus clearance.

BACKGROUND AND OBJECTIVES: The increasing demand for intravenous immunoglobulin (IVIG) necessitates the development of improved plasma fractionation methods, providing higher immunoglobulin G (IgG) recovery. Here, we describe a new IVIG production process resulting in a high yield of IgG and effective reduction of physico-chemically resistant viruses. MATERIALS AND METHODS: IgG was purified from Cohn fraction II+III by caprylic acid treatment, polyethylene glycol precipitation, anion-exchange chromatography, nanofiltration and ultrafiltration. Stability of the purified IgG was studied in different formulations. Virus reduction was studied with two viruses: bovine viral diarrhoea virus, assessed by an infectivity assay; and human parvovirus B19, assessed by polymerase chain reaction. RESULTS: The combination of caprylic acid treatment with polyethylene glycol precipitation and a single anion-exchange chromatography yielded polymer-free, pure IgG. The purified IgG could be filtered through a small pore-size virus filter (Millipore V-NFP) with high throughput and excellent yield. The formulated product was stable as a 100 g/l IgG solution. Bovine viral diarrhoea virus was effectively inactivated by the caprylic acid treatment, and parvovirus B19 was effectively removed in the polyethylene glycol precipitation and nanofiltration stages, the total reduction of parvovirus being approximately 14 log10. CONCLUSIONS: The new process gives pure and stable IgG solution with an average yield of 4.8 g of IgG per kg of recovered plasma and has a very high capacity to remove even physico-chemically resistant viruses.

Viral safety of Nanogam, a new 15 nm-filtered liquid immunoglobulin product.

BACKGROUND AND OBJECTIVES: Producers of plasma derivatives continuously improve the viral safety of their products by, for example, introducing additional virus-reducing steps into the manufacturing process. Here we present virus-elimination studies undertaken for a number of steps employed in a new manufacturing process for liquid intravenous immunoglobulin (Nanogam) that comprises two specific virus-reducing steps: a 15-nm filtration step combined with pepsin treatment at pH 4.4 (pH 4.4/15NF); and solvent-detergent (SD) treatment. The manufacturing process also includes precipitation of Cohn fraction III and viral neutralization, which contribute to the total virus-reducing capacity of the manufacturing process. In addition, the mechanism and robustness of the virus-reducing steps were studied. MATERIALS AND METHODS: Selected process steps were studied with spiking experiments using a range of lipid enveloped (LE) and non-lipid-enveloped (NLE) viruses. The LE viruses used were bovine viral diarrhoea virus (BVDV), human immunodeficiency virus (HIV) and pseudorabies virus (PRV); the NLE viruses used were parvovirus B19 (B19), canine parvovirus (CPV) and encephalomyocarditis virus (EMC). After spiking, samples were collected and tested for residual infectivity, and the reduction factors were calculated. For B19, however, removal of B19 DNA was measured, not residual infectivity. To reveal the contribution of viral neutralization, bovine parvovirus (BPV) and hepatitis A virus (HAV) were used. RESULTS: For the pH 4.4/15NF step, complete reduction (> 6 log(10)) was demonstrated for all viruses, including B19, but not for CPV (> 3.4 but < or = 4.2 log(10)). Robustness studies of the pH 4.4/15NF step with CPV showed that pH was the dominant process parameter. SD treatment for 10 min resulted in complete inactivation (> 6 log(10)) of all LE viruses tested. Precipitation of Cohn fraction III resulted in the significant removal (3-4 log(10)) of both LE and NLE viruses. Virus-neutralization assays of final product revealed significant reduction (> or = 3 log(10)) of both BPV and HAV. CONCLUSIONS: The manufacturing process of Nanogam comprises two effective steps for the reduction of LE viruses and one for NLE viruses. In addition, the precipitation of Cohn fraction III and the presence of neutralizing antibodies contribute to the total virus-reducing capacity of Nanogam. The overall virus-reducing capacity was > 15 log(10) for LE viruses. For the NLE viruses B19, CPV and EMC, the overall virus-reducing capacities were > 10, > 7 and > 9 log(10), respectively. Including the contribution of immune neutralization, the overall virus-reducing capacity for B19 and HAV is estimated to be > 10 log(10).

Development of a highly purified multicomponent leukocyte IFN-alpha product.

A purification process was developed to obtain a human interferon- alpha (IFN-alpha) product that contains all major IFN-alpha subtypes produced by human leukocytes. The purification was accomplished by immunoaffinity chromatography using two monoclonal antibodies (mAb) and gel filtration. The process comprised two effective virus inactivation steps, solvent detergent treatment, and incubation at low pH, and the purified product was filtered with a 15-nm pore size virus removal filter. The overall yield of IFN-alpha in the process was about 60% when starting from the culture supernatant of Sendai virus-induced human leukocytes. The specific activity was about 1.0 x 10(8) IU/mg. The level of DNA and protein impurities including mouse IgG was very low. The product contained seven main subtypes: IFN-alpha 1, IFN-alpha 2, IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 17, and IFN-alpha 21. The subtypes IFN-alpha 4 and IFN-alpha 7 were minor components. Reverse-phase HPLC indicated a constant subtype composition for the product from batch to batch. Stabilization of the pure IFN-alpha solution with albumin and Tween 80 was compared. In virus filtration, a better yield and higher filtration capacity were obtained with Tween. The addition of albumin resulted in the formation of IFN-albumin aggregates. During long-term storage, IFN-alpha was stable in both solutions for 2 years at 2-8 degrees C. The new method makes it possible to extensively purify all major IFN-alpha subtypes and obtain a virus-safe and stable product with a constant subtype composition.

Development of a pharmaceutical apotransferrin product for iron binding therapy.

High-dose chemotherapy of patients with haematological malignancies results in extracellular iron accumulation and appearance of non-transferrin-bound iron, which is thought to predispose the patients to septic infections and contribute to organ toxicity. We describe the development of a human plasma-derived apotransferrin product for iron binding therapy. The product is purified from Cohn fraction IV of human plasma by two ion exchange chromatography steps and ultrafiltration. The process comprises solvent detergent treatment as the main virus inactivation step and 15 nm virus filtration and polyethylene glycol precipitation as removal steps for physico-chemically resistant infectious agents. Product characterization by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. The purity of the product was more than 98%, main impurities being IgG, IgA and hemopexin. The product had intact iron binding capacity and native conformation. A stable liquid formulation for the finished product was developed. The product has proved safe and well tolerated in early clinical trials in iron binding therapy.

Leukocyte interferon-alpha in the treatment of chronic hepatitis C in Finland.

BACKGROUND: To evaluate the efficacy of leukocyte interferon in previously untreated patients with chronic hepatitis C, 97 patients were enrolled in a prospective study in Finland with a stepped-care management protocol. METHODS: The treatment was initiated with 3 million units of interferon-alpha subcutaneously three times a week. At 3 months, if the serum alanine aminotransferase was still abnormal, the dose was doubled. If serum hepatitis C virus (HCV) RNA had turned negative at 6 months, the treatment was stopped; if it was still positive, treatment was continued for up to 12 months. All patients were followed up after treatment for 6 months. Altogether, 74 patients completed the treatment and follow-up periods. RESULTS: Of all the originally enrolled patients 36% (35 of 97) achieved sustained virologic response, defined as HCV RNA negativity 6 months after the end of treatment. The commonest HCV genotype among these patients was 3a, and as many as 52% of such patients achieved sustained virologic response. Thirty-two per cent of the patients had HCV genotype 1a, 1b, or a mixture of these; a sustained response was achieved in only 6% of such patients but in 50% of patients with a non-1 genotype. Adverse effects caused treatment cessation for 10% of the patients and IFN dose reduction for 20%. CONCLUSIONS: Monotherapy with human leukocyte interferon resulted in sustained virologic response in 36% of patients with chronic hepatitis C. In those infected with a HCV genotype other than 1, the sustained virologic response rate was 50%.

Structural characterisation of N-linked and O-linked oligosaccharides derived from interferon-alpha2b and interferon-alpha14c produced by Sendai-virus-induced human peripheral blood leukocytes.

We have previously isolated and partially characterised the components of a highly purified interferon-alpha (IFN-alpha) preparation produced by Sendai-virus-induced human peripheral blood leukocytes. Nine IFN-alpha species were identified, and two of these were found to be glycosylated [Nyman, T. A., Tölö, H., Parkkinen, J. & Kalkkinen, N. (1998) Identification of nine interferon-alpha subtypes produced by Sendai-virus-induced human peripheral blood leukocytes, Biochem. J. 329, 295-302]. Here, we isolated the N-linked oligosaccharides of IFN-alpha14c and the O-linked chains of IFN-alpha2b, and the glycans were characterised by electrospray tandem mass spectrometry and by specific glycosidase digestions monitored by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The IFN-alpha14c N-glycans were shown to exhibit core-fucosylated biantennary glycans, with about 10% carrying an additional alpha1,3-linked fucose unit at the antennae. The IFN-alpha2b was shown to carry about 50% core type-1 disialyltetrasaccharides, 30% core type-1 monosialyltrisaccharides and 20% core type-2 monosialylpentasaccharides.

Conjugation of interferon alpha to a humanized monoclonal antibody (HuBrE-3vl) enhances the selective localization and antitumor effects of interferon in breast cancer xenografts.

Human mammary carcinoma xenografts (MCF-7) growing in nude mice were treated with natural interferon alpha (n-IFN-alpha) alone or conjugated to a humanized monoclonal antibody (MoAb) anti-breast mucin (HuBrE-3vl) or to irrelevant human IgG1kappa. The IFN and the conjugates were administered as 20 intra-lesional (i.l.) injections to 1 of 2 xenografts in each mouse, or i.p. The growth inhibitory effects of HuBrE-3vl/nIFN-alpha were significantly greater than those of nIFN-alpha used as a single agent or conjugated to HuIgG1kappa. These effects occurred locally in the tumors receiving i.l. injections and systemically, although to a slightly lesser extent, in the noninjected tumors of mice treated i.l. and in the xenografts of mice treated i.p. Biodistribution studies showed that the uptake of 125I-HuBrE-3vl/nIFN-alpha by the tumors 24 hours after i.l. or s.c. injection was greater than that of 125I-HuIgG1kappa/nIFN-alpha, 125I-nIFN-alpha alone, or by normal tissues, documenting a tumor targeting effect and favorable tumor:normal tissues (T:NT) ratios. The targeting effects and the resulting tumor growth inhibition were favored by the IFN-mediated up-regulation of the HuBrE-3vl reactive antigen, which was more prominent after 3 weeks of treatment with HuBrE-3vl/nIFN-alpha. These results were superior to those we obtained previously with nIFN-alpha conjugated to another MoAb of the same group (Mc5). These studies point out the potential usefulness of HuBrE-3vl/nIFN-alpha for the local and systemic treatment of breast cancer lesions by providing a means of delivering high doses of IFN to the tumors while minimizing the amount of IFN binding to normal tissues.

Identification of nine interferon-alpha subtypes produced by Sendai virus-induced human peripheral blood leucocytes.

The human interferon-alpha (IFN-alpha) family is encoded by 13 different functional genes, and including all cloned sequence variants there are 28 potential IFN-alpha proteins. To find out which of the described sequences are expressed in normal human leucocytes, we have isolated and partly characterized the components of a highly purified IFN-alpha preparation produced by Sendai virus-induced human peripheral blood leucocytes. The identification protocol consisted of N-terminal sequencing and mass mapping of the proteins separated by reverse-phase HPLC and/or SDS/PAGE. The highly purified leucocyte IFN-alpha preparation was found to contain at least nine different IFN-alpha species: IFN-alpha1a, IFN-alpha2b, IFN-alpha4b, IFN-alpha7a, IFN-alpha8b, IFN-alpha10a, IFN-alpha14c, IFN-alpha17b and IFN-alpha21b. IFN-alpha1a was the major subtype, comprising approx. 30% of total leucocyte IFN-alpha. IFN-alpha14c, the only subtype containing potential N-glycosylation sites, was shown to be glycosylated at Asn-72. Molecular mass determination of the intact proteins by electrospray ionization MS showed that there are no other post-translational modifications in the IFN-alpha subtypes than the glycosylation of IFN-alpha2b and IFN-alpha14c. Only one sequence variant was found for each subtype, suggesting that the other described gene sequences represent allelic variants or mutations that are more rarely found in the general population.

Biosynthesis and N-glycosylation of human interferon-gamma. Asn25 and Asn97 differ markedly in how efficiently they are glycosylated and in their oligosaccharide composition.

Interferon-gamma (IFN-gamma) is a secretory glycoprotein produced by T cells in response to antigenic or mitogenic stimuli. We studied the kinetics of the synthesis, N-glycosylation, and secretion of IFN-gamma in human CD8+ T lymphocytes stimulated via T-cell receptor. Highly elevated IFN-gamma mRNA levels were found as early as 1 h after stimulation. Maximal IFN-gamma protein synthesis was observed 2-4 h after induction and appeared to correlate to steady-state IFN-gamma mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN-gamma from T cells was very rapid, the secretion half-time being approximately 20-25 min. Inhibition of N-glycosylation by tunicamycin dramatically reduced the expression of IFN-gamma, but did not block its secretion. Natural IFN-gamma is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell-free translation/glycosylation system with mutated IFN-gamma constructs lacking either one of the potential glycosylation sites suggested that Asn25 is more efficiently glycosylated than Asn97. Site-specific oligosaccharide analysis of natural IFN-gamma by glycosidase treatment followed by matrix-assisted-laser-desorption-ionization mass spectrometry revealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Asn25 consisted of fucosylated, mainly complex-type oligosaccharides, whereas the glycans at Asn97 were more heterogeneous, with hybrid and high-mannose structures. Our results emphasize the essential role of N-linked glycans in the biology of IFN-gamma and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.

Long-term follow-up of anti-IgA antibodies in healthy IgA-deficient adults.

A follow-up study of anti-IgA antibodies in 159 healthy blood donors with severe deficiency of serum IgA (< 0.05 mg/L) and in 45 donors with decreased serum IgA levels (0.05-799 mg/L), identified in 1971-1980, was carried out. Initially anti-IgA antibodies were determined by a hemagglutination (HA) method and two reexaminations were done in 1990-1992 by an enzyme immunoassay. The median follow-up period was 19 years, during which anti-IgA level was changed considerably in only four persons, increased in two, and high level antibodies (> 1/1000 by HA) appeared in two. In reexaminations anti-IgA antibodies were found in 30 (19%) subjects with severe IgA deficiency and the antibody levels remained relatively constant in those who had high and medium antibody levels. Anti-IgA antibodies were not found in subjects with decreased, but detectable serum IgA. Thus it seems that only those healthy adults who have severe IgA deficiency develop anti-IgA antibodies and their anti-IgA levels remain fairly constant. Of the 159 subjects with severe IgA deficiency, 66 had a history of IgA exposure, but no correlation to anti-IgA development was noted.

An enzyme immunoassay for the determination of anti-IgA antibodies using polyclonal human IgA.

An enzyme immunoassay (EIA) for screening and quantitation of serum anti-IgA antibodies of IgG class is described. This method is based on the use of purified polyclonal human serum IgA as the coating antigen and a commercial alkaline phosphatase-conjugated anti-human IgG as the detecting antibody. Nonspecific reactions were minimized by blocking vacant protein binding sites with bovine serum albumin and by using individual sample blanks. The IgA specificity of a positive antibody finding was confirmed by testing inhibition: pooled normal human serum inhibited the binding of specific antibodies by over 80%. The same degree of inhibition could also be demonstrated by a commercial myeloma IgA preparation and by the IgA used for coating but not by IgA-deficient serum (< 0.05 mg/l). On the basis of the mean anti-IgA antibody titre in EIA, a value of 12,000 arbitrary units of anti-IgA per litre (AU/l) was assigned to a patient serum used as standard in the assay. Anti-IgA results obtained by EIA and haemagglutination correlated well, which makes it possible to compare earlier HA results with those obtained now by EIA. The measuring range of the assay was 0.6-27 AU/l and the lowest quantifiable concentration 7 AU/l. The dilution requirement for serum was 1/16. The interassay coefficients of variation for control sera with antibody levels from 35 AU/l to 3770 AU/l varied from 9 to 12%.

Long-term persistence of selective IgA deficiency in healthy adults.

A follow-up study of 204 healthy blood donors with IgA deficiency, identified between 1971 and 1980, was carried out. Sera were initially screened by a double diffusion method and 182 were retested by a more sensitive haemagglutination inhibition method. A reexamination was performed in 1990 and, again, in 1991-1992 using an enzyme immunoassay (EIA) developed for the measurement of very low concentrations of IgA. The median follow-up period was 19 years, and in 159 (78%) subjects no serum IgA could be detected in any of the measurements. In 42 (21%) subjects, serum IgA was detectable (> 0.18 mg/L), but the level was below the lower limit of the reference range for adults (800 mg/L) and remained relatively constant. Three subjects showed minute amounts of IgA by EIA (0.2-3 mg/L) in one of the follow-up samples in 1990-1992, but the level was below the detection limit of the EIA (0.05 mg/L) in the other sample. Thus, not only does primary IgA deficiency appear to be permanent, but also lower than normal serum IgA levels remain the same in healthy adults.

A sensitive enzyme immunoassay for the measurement of low concentrations of IgA.

A microtitre plate enzyme immunoassay (EIA) for determining low concentrations of IgA is described and validated for serum and plasma products. The measuring range of the assay was 3.3-150 micrograms/l. The predilution requirement of samples was matrix dependent, ranging from 1/16 for serum to 1/2 for 4% albumin. Dilute protein solutions required no predilution. The limits of detection were 50 micrograms/l for serum, 25 micrograms/l for intravenous immunoglobulin, 13 micrograms/l for 20% albumin, 7 micrograms/l for 4% albumin and 3 micrograms/l for washing solutions of blood cell components. Interassay coefficients of variation over the range of 3.4 mg to 1.5 g IgA/l ranged from 3.8 to 5.7%. Respective values for two low-level sera, containing 309 and 512 micrograms IgA/l, were 15.5% and 11.1%. Comparison of the EIA with a commercial radial immunodiffusion (RID) method showed that the results of the two assays correlated well ([EIA] = 0.877 x [RID] + 0.401 mg/l, r = 0.996, n = 20). This assay is also suitable for the large-scale screening of blood donors.

Determination of anti-IgA antibodies with a flow cytometer-based microbead immunoassay (MIA).

A flow cytometer-based microbead immunoassay (MIA) was employed to detect anti-IgA antibodies in patients with IgA deficiency. 3 microns latex particles were coated with purified IgA and serum anti-IgA antibodies of the IgG class were detected with FITC-conjugated anti-human IgG. Antibodies against three different IgA preparations were tested from 22 patients samples as well as 20 controls and compared with a conventional enzyme-linked immunosorbent assay (ELISA) and a passive hemagglutination assay (HA). There was a very close correlation between the results obtained with the MIA and the ELISA assay and between MIA and the HA. Because of the low intra-assay variation and good linearity of the assay, the analysis of one single serum dilution was sufficient to determine the anti-IgA level of a patient and no titration series was required. We conclude that MIA is a satisfactory alternative method for routine anti-IgA antibody determinations. For laboratories already equipped with a flow cytometer the assay is cost effective.

An optimized assay for prekallikrein activator activity in human plasma products.

Prekallikrein activator (PKA) assay is described. PKA was measured indirectly by allowing it to generate kallikrein from prekallikrein and then measuring the amidolytic activity of kallikrein by using the synthetic chromogenic substrate S-2302. It was ensured that sufficient amounts of substrates were included in both enzymatic phases of the assay, and the pH and ionic strength which seemed to be very important in the assay were carefully controlled. Both a kinetic assay and a simple endpoint assay are described. Other amidolytic enzymes probably present in plasma products are also estimated and a correction is made to exclude their role in the assay result. The precision of the kinetic assay is characterized by the coefficient of variation of 3.1% within an assay and the reproducibility by that of 4.8% between assays. For the endpoint assay both values are 4.8%.