Multivariate analysis applied to complex biological medicines.

A biological medicine (or biologicals) is a term for a medicinal compound that is derived from a living organism. By their very nature, they are complex and often heterogeneous in structure, composition and biological activity. Some of the oldest pharmaceutical products are biologicals, for example insulin and heparin. The former is now produced recombinantly, with technology being at a point where this can be considered a defined chemical entity. This is not the case for the latter, however. Heparin is a heterogeneous polysaccharide that is extracted from the intestinal mucosa of animals, primarily porcine, although there is also a significant market for non-porcine heparin due to social and economical reasons. In 2008 heparin was adulterated with another sulfated polysaccharide. Unfortunately this event was disastrous and resulted in a global public health emergency. This was the impetuous to apply modern analytical techniques, principally NMR spectroscopy, and multivariate analyses to monitor heparin. Initially, traditional unsupervised multivariate analysis (principal component analysis (PCA)) was applied to the problem. This was able to distinguish animal heparins from each other, and could also separate adulterated heparin from what was considered bona fide heparin. Taught multivariate analysis functions by training the analysis to look for specific patterns within the dataset of interest. If this approach was to be applied to heparin, or any other biological medicine, it would have to be taught to find every possible alien signal. The opposite approach would be more efficient; defining the complex heterogeneous material by a library of bona fide spectra and then filtering test samples with these spectra to reveal alien features that are not consistent with the reference library. This is the basis of an approach termed spectral filtering, which has been applied to 1D and 2D-NMR spectra, and has been very successful in extracting the spectral features of adulterants in heparin, as well as being able to differentiate supposedly biosimilar products. In essence, the filtered spectrum is determined by subtracting the covariance matrix of the library spectra from the covariance matrix of the library spectra plus the test spectrum. These approaches are universal and could be applied to biological medicines such as vaccine polysaccharides and monoclonal antibodies.

Anti-Factor B and Anti-C3b Autoantibodies in C3 Glomerulopathy and Ig-Associated Membranoproliferative GN.

In C3 glomerulopathy (C3G), the alternative pathway of complement is frequently overactivated by autoantibodies that stabilize the C3 convertase C3bBb. Anti-C3b and anti-factor B (anti-FB) IgG have been reported in three patients with C3G. We screened a cohort of 141 patients with C3G and Ig-associated membranoproliferative GN (Ig-MPGN) for anti-FB and anti-C3b autoantibodies using ELISA. We identified seven patients with anti-FB IgG, three patients with anti-C3b IgG, and five patients with anti-FB and anti-C3b IgG. Of these 15 patients, ten were diagnosed with Ig-MPGN. Among those patients with available data, 92% had a nephrotic syndrome, 64% had AKI, and 67% had a documented infection. Patients negative for anti-C3b and anti-FB IgG had much lower rates of infection (17 [25%] patients with C3G and one [10%] patient with Ig-MPGN). After 48 months, four of 15 (26%) positive patients had developed ESRD or died. All 15 patients had high plasma Bb levels, six (40%) patients had low levels of C3, and nine (60%) patients had high levels of soluble C5b9. In vitro, IgG purified from patients with anti-FB Abs selectively enhanced C3 convertase activity; IgG from patients with anti-C3b/anti-FB Abs enhanced C3 and C5 cleavage. IgG from patients with anti-C3b Abs stabilized C3bBb and perturbed C3b binding to complement receptor 1 but did not perturb binding to factor H. In conclusion, the prevalence of anti-C3b/anti-FB Abs and alternative pathway activation is similar in Ig-MPGN and C3G, suggesting similar pathogenic mechanisms. Identification of the underlying defect in Ig-MPGN could lead to improved treatment.

C5 nephritic factors drive the biological phenotype of C3 glomerulopathies.

C3 Glomerulopathies, which include Dense Deposit Disease and C3 Glomerulonephritis, are associated with genetic and acquired dysregulation of the C3 convertase alternative pathway of complement. The potential role of the activation of the C5 convertase has not been studied extensively. Here we analyzed IgG samples from patients with C3 Glomerulopathies to identify circulating autoantibodies that stabilize the C3 alternative pathway (C3 Nephritic Factors) as well as C5 convertases (C5 Nephritic Factors), thus preventing decay of these enzyme complexes. Rare variants in alternative pathway genes were found in 28 of 120 tested patients. C3 and C5 Nephritic Factors were found in 76 of 101 (75%) and 29 of 59 (49%) of the patients, respectively. Therefore, we compared the results of the assays for the C3 and C5 nephritic factors functional activity: 29% were positive for C3 Nephritic Factors alone, 39% were positive for both C3 and C5 Nephritic Factors, and 10% were positive for C5 Nephritic Factors alone. We found that the addition of properdin-enhanced stabilization of C3 convertase in the presence of IgG doubly positive for both Nephritic Factors, while it did not modify the stabilization mediated by IgG solely positive for C3 Nephritic Factors. Both C3 and C5 Nephritic Factors correlated with C3 consumption, while only C5 Nephritic Factors correlated with sC5b9 levels. C5 Nephritic Factors-positive patients were more likely to have C3 Glomerulonephritis than Dense Deposit Disease. Thus, dysregulation of the C5 convertase contributes to C3 Glomerulopathies inter-disease differences and may have direct therapeutic implications.

A Familial C3GN Secondary to Defective C3 Regulation by Complement Receptor 1 and Complement Factor H.

C3 glomerulopathy is a recently described form of CKD. C3GN is a subtype of C3 glomerulopathy characterized by predominant C3 deposits in the glomeruli and is commonly the result of acquired or genetic abnormalities in the alternative pathway (AP) of the complement system. We identified and characterized the first mutation of the C3 gene (p. I734T) in two related individuals diagnosed with C3GN. Immunofluorescence and electron microscopy studies showed C3 deposits in the subendothelial space, associated with unusual deposits located near the complement receptor 1 (CR1)-expressing podocytes. In vitro, this C3 mutation exhibited decreased binding to CR1, resulting in less CR1-dependent cleavage of C3b by factor 1. Both patients had normal plasma C3 levels, and the mutant C3 interacted with factor B comparably to wild-type (WT) C3 to form a C3 convertase. Binding of mutant C3 to factor H was normal, but mutant C3 was less efficiently cleaved by factor I in the presence of factor H, leading to enhanced C3 fragment deposition on glomerular cells. In conclusion, our results reveal that a CR1 functional deficiency is a mechanism of intraglomerular AP dysregulation and could influence the localization of the glomerular C3 deposits.

Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin.

Because of the complexity and global nature of the heparin supply chain, the control of heparin quality during manufacturing steps is essential to ensure the safety of the final active pharmaceutical ingredient (API). For this reason, there is a need to develop consistent analytical methods able to assess the quality of heparin early in production (i.e., as the crude heparin before it is purified to API under cGMP conditions). Although a number of analytical techniques have been applied to characterize heparin APIs, few of them have been applied for crude heparin structure and composition analyses. Here, to address this issue, NMR spectroscopy and chemometrics were applied to characterize 88 crude heparin samples. The samples were also analyzed by strong anion exchange HPLC (SAX-HPLC) as an orthogonal check of the purity levels of the crudes analyzed by NMR. The HPLC data showed that the chemometric analysis of the NMR data differentiated the samples based on their purity. These orthogonal approaches differentiated samples according their glycosaminoglycan (GAG) composition and their mono and disaccharide composition and structure for each GAG family (e.g., heparin/heparan, dermatan sulfate, and chondroitin sulfate A). Moreover, quantitative HSQC and multivariate analysis (PCA) were used to distinguish between crude heparin of different animal and tissue sources.

Complement factor B mutations in atypical hemolytic uremic syndrome-disease-relevant or benign?

Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association.

Antiproliferative effect of ASC-J9 delivered by PLGA nanoparticles against estrogen-dependent breast cancer cells.

Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of the most popular polymeric devices for chemotherapeutic-based nanoformulations against several kinds of malignant diseases. Promising properties, including long-circulation time, enhanced tumor localization, interference with "multidrug" resistance effects, and environmental biodegradability, often result in an improvement of the drug bioavailability and effectiveness. In the present work, we have synthesized 1,7-bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (ASC-J9) and developed uniform ASC-J9-loaded PLGA nanoparticles of about 120 nm, which have been prepared by a single-emulsion process. Structural and morphological features of the nanoformulation were analyzed, followed by an accurate evaluation of the in vitro drug release kinetics, which exhibited Fickian law diffusion over 10 days. The intracellular degradation of ASC-J9-bearing nanoparticles within estrogen-dependent MCF-7 breast cancer cells was correlated to a time- and dose-dependent activity of the released drug. A cellular growth inhibition associated with a specific cell cycle G2/M blocking effect caused by ASC-J9 release inside the cytosol allowed us to put forward a hypothesis on the action mechanism of this nanosystem, which led to the final cell apoptosis. Our study was accomplished using Annexin V-based cell death analysis, MTT assessment of proliferation, radical scavenging activity, and intracellular ROS evaluation. Moreover, the intracellular localization of nanoformulated ASC-J9 was confirmed by a Raman optical imaging experiment designed ad hoc. PLGA nanoparticles and ASC-J9 proved also to be safe for a healthy embryo fibroblast cell line (3T3-L1), suggesting a possible clinical translation of this potential nanochemotherapeutic to expand the inherently poor bioavailability of hydrophobic ASC-J9 that could be proposed for the treatment of malignant breast cancer.

C3 nephritic factor associated with C3 glomerulopathy in children.

BACKGROUND: C3 glomerulopathy (C3G) is characterized by predominant C3 deposits in glomeruli and dysregulation of the alternative pathway of complement. Half of C3G patients have a C3 nephritic factor (C3NeF). C3G incorporated entities with a range of features on microscopy including dense deposit diseases (DDD) and C3 glomerulonephritis (C3GN). The aim of this work was to study children cases of C3G associated with C3NeF. METHODS: We reviewed 18 cases of C3G with a childhood onset associated with C3NeF without identified mutations in CFH, CFI, and MCP genes. RESULTS: Clinical histories started with recurrent hematuria for seven patients, nephrotic syndrome for four, acute post-infectious glomerulonephritis for three and acute renal failure for four. Twelve patients had a low C3 at first investigation. Kidney biopsy showed ten C3GN and eight DDD. Twenty-three percent of the patients tested presented elevated sC5b9. Seven patients relapsed 3 to 6 years after the onset. At the end of follow-up, two patients were under dialysis, 11 had a persistent proteinuria, five had none; four patients did not follow any treatment. Steroids were first used in 80 % of cases. CONCLUSIONS: C3NeF associated C3G has a heterogeneous presentation and outcome. Anti-proteinuric agents may control the disease during follow-up, even after nephrotic syndrome at the onset. The efficiency of immunosuppressive therapy remains questionable.

WITHDRAWN: Erratum to "Increasing regular donors through a psychological approach which reduces the onset of vasovagal reactions" [Transfus. Apheresis Sci. 47 (3) (2012) 301-304].

This article has been withdrawn at the request of the author. The Publisher apologizes for any inconvenience this may cause.

Giving blood donors something to drink before donation can prevent fainting symptoms: is there a physiological or psychological reason?

The vasovagal reaction has been widely studied but its anatomic and physiological nature remains uncertain. The mechanisms underlying vasovagal reaction related to blood donation are not completely understood either. Does its occurrence depend on the blood donors' physical characteristics and health variables or psychological factors? On the basis that a psychological approach considerably prevents donor reactions, the effect of fruit juice ingestion was studied in a group of 1849 first-time high-school students as a simple strategy to avoid systemic reactions at blood donation. The reasons for the psychological effect of this hydration protocol are stressed also in light of previous physiological studies on the hemodynamic effects of water or carbohydrate drinks.

Emotional support to apheresis donors: effect and implication.

It is widely accepted that well-being and satisfaction are unavoidably interlinked requirements that predispose donors to become regular. Detecting in advance even the mildest symptoms of discomfort during first donation, that may simply be the result of an unknown experience, is useful in preventing more disturbing symptoms (mens sana in corpore sano). Being able to recognize any form of uneasiness, even when not immediately apparent, creates in donors the awareness that staff is professionally skilled and really interested in their safety and comfort. This reinforces the staff/donor relationship and gives donors the chance to keep their calm and to learn how to manage disturbing emotions. Emotional support given to first-time platelet familiar donors during donation prevented or mitigated the onset of vaso-vagal symptoms and also freed positive emotions that reinforced the desire to donate. As a result, 62% of these donors, informed about the shortage of voluntary platelet donors, spontaneously gave their address contact to be called also for non-familiar recipients. The application of the psychological approach in Italy increased the rate of donor return to 97% and its partial application in Serbia was useful in converting familiar donors into voluntary ones.

Increasing regular donors through a psychological approach which reduces the onset of vasovagal reactions.

Vasovagal reactions, even in their mildest manifestations, deter donors from becoming regulars. They mainly occur during the first donation. A psychological approach based on a path involving three steps (welcome, psychological interview and accompanying the donor to the donation site) was performed in a group of 387 first-time donors. As a result, 96.64% of them became regular donors, 0.77% had slight vasovagal symptoms such as paleness or a feeling of weakness. Complying with the requirement and expectations of donors has proven to be an effective strategy in dissipating fears of donation and avoiding vasovagal symptoms and thus influenced their return.

Blood donor satisfaction and the weak link in the chain of donation process.

Recruiting and retaining enough donors is a problem. Six hundred and thirty nine blood donors were interviewed to identify the elements that can influence their satisfaction and the decision to become regulars. The majority of them were satisfied with staff skill and communication, they declared they were calm before donating and felt well after donation. First-time donors (FTDs) were more frightened, showed anticipatory anxiety, were less sure they would donate again (p<0.01) and the youngest were also less satisfied with the staff's behavior after donation. Identifying the more scared FTDs and diminishing their anxiety before donating could positively influence their decision to become regulars.

NMR spectroscopy and chemometric models to detect a specific non-porcine ruminant contaminant in pharmaceutical heparin.

Heparin has been used successfully as a clinical antithrombotic for almost one century. Its isolation from animal sources (mostly porcine intestinal mucosa) involves multistep purification processes starting from the slaughterhouse (as mucosa) to the pharmaceutical plant (as the API). This complex supply chain increases the risk of contamination and adulteration, mainly with non-porcine ruminant material. The structural similarity of heparins from different origins, the natural variability of the heparin within samples from each source as well as the structural changes induced by manufacturing processes, require increasingly sophisticated methods capable of detecting low levels of contamination. The application of suitable multivariate classification approaches on API 1H NMRspectra serve as rapid and reliable tools for product authentication and the detection of contaminants. Soft Independent Modeling of Class Analogies (SIMCA), Discriminant Analysis (DA), Partial Least Square Discriminant Analysis (PLS-DA) and local classification methods (kNN, BNN and N3) were tested on about one hundred certified heparin samples produced by 14 different manufacturers revealing that Partial Least Squares Discriminant Analysis (PLS-DA) provided the best discrimination of contaminated batches, with a balanced accuracy of 97%.

Genotypic/phenotypic patterns of HIV-1 integrase resistance to raltegravir.

OBJECTIVES: To understand the dynamic viral evolution observed during failure on raltegravir-containing regimens, we studied the genotypic and phenotypic patterns of resistance to raltegravir and the residual replication capacity (rRC) of HIV-1 variants selected in vivo. METHODS: Clonal genotypic analyses were performed on sequential HIV-1 integrase sequences amplified from 11 failing patients and sampled every 4-24 weeks for up to 64 weeks. Fully replicating recombinant viruses were generated using modified vectors in which selected viral integrase genes amplified from patients' plasma were cloned. rRC was measured by a novel multiple cycle competition assay. Resistance to raltegravir and the rRC of resistant HIV-1 variants selected in vivo were evaluated in purified CD4+ T cells. RESULTS: In all of the patients but one, failure was associated with selection of mutations in positions 143, 148 or 155. Unlike mutations at position 143 (Y143S/K/R), identified alone or in combination with others, mutations at position 148 and 155 were always found in combination. A wide range of resistance levels to raltegravir [from 10- to 770-fold change in 50% inhibitory concentration (IC(50)) compared with baseline] was observed using recombinant viral clones. Finally, rRC was not significantly altered in highly resistant variants. DISCUSSION: Two patterns of viral evolution were observed in the resistant viral populations, driving the variants towards a fast (most of them with G140S + Q148H mutations) or progressive increase in resistance to raltegravir. These results may have implications either for the evaluation of genotypic results, or for the correct clinical use of the compound.

Molecular epidemiology of KI and WU polyomaviruses in infants with acute respiratory disease and in adult hematopoietic stem cell transplant recipients.

Polyomaviruses KI (KIPyV) and WU (WUPyV) were described recently in children with acute respiratory disease. The pathogenic potential of these human viruses has not been determined completely, but a correlation between immunosuppression and virus reactivation has been suggested. In the present study, the association between KI/WUPyV infection and immunosuppression was investigated using sequential nasopharyngeal aspirates from asymptomatic adult hematopoietic stem cell transplant recipients. In parallel, an investigation on the WU/KIPyV prevalence in children with acute respiratory disease was also carried out. Two of the 126 samples obtained from the 31 hematopoietic transplant recipients were positive for KIPyV (1 sample, 0.79%) and WUPyV (1 sample, 0.79%). Both samples were obtained 15 days after allogeneic transplantation and virus persistence was not observed in subsequent samples. In symptomatic children, 7 of the 486 nasopharyngeal aspirates were positive for WUPyV (1.4%) and 1 for KIPyV (0.2%). Single polyomavirus infection was detected in four patients, whereas the remaining patients were co-infected with respiratory syncityal virus (three patients) or adenovirus (one patient). The results suggest that WU/KIPyVs have a limited circulation in Italy and a low pathogenic potential in young children. Brief and asymptomatic infection can occur in hematopoietic transplant recipients.

Muscle Oxygen Delivery in the Forearm and in the Vastus Lateralis Muscles in Response to Resistance Exercise: A Comparison Between Nepalese Porters and Italian Trekkers.

Altitude ascending represents an intriguing experimental model reproducing physiological and pathophysiological conditions sharing hypoxemia as the denominator. The aim of the present study was to investigate fractional oxygen extraction and blood dynamics in response to hypobaric hypoxia and to acute resistance exercises, taking into account several factors including different ethnic origin and muscle groups. As part of the "Kanchenjunga Exploration & Physiology" project, six Italian trekkers and six Nepalese porters took part in a high altitude trek in the Himalayas. The measurements were carried out at low (1,450 m) and high altitude (HA; 4,780 m). Near-infrared spectroscopy (NIRS)-derived parameters, i.e., Tot-Hb and tissue saturation index (TSI), were gathered at rest and after bouts of 3-min resistive exercise, both in the quadriceps and in the forearm muscles. TSI decreased with altitude, particularly in forearm muscles (from 66.9 to 57.3%), whereas the decrement was less in the quadriceps (from 62.5 to 57.2%); Nepalese porters were characterized by greater values in thigh TSI than Italian trekkers. Tot-Hb was increased after exercise. At altitude, such increase appeared to be higher in the quadriceps. This effect might be a consequence of the long-term adaptive memory due to the frequent exposures to altitude. Although speculative, we suggest a long-term adaptation of the Nepalese porters due to improved oxygenation of muscles frequently undergoing hypoxic exercise. Muscle structure, individual factors, and altitude exposure time should be taken into account to move on the knowledge of oxygen delivery and utilization at altitude.

Dynamic features of the selective pressure on the human immunodeficiency virus type 1 (HIV-1) gp120 CD4-binding site in a group of long term non progressor (LTNP) subjects.

The characteristics of intra-host human immunodeficiency virus type 1 (HIV-1) env evolution were evaluated in untreated HIV-1-infected subjects with different patterns of disease progression, including 2 normal progressor [NP], and 5 Long term non-progressor [LTNP] patients. High-resolution phylogenetic analysis of the C2-C5 env gene sequences of the replicating HIV-1 was performed in sequential samples collected over a 3-5 year period; overall, 301 HIV-1 genomic RNA sequences were amplified from plasma samples, cloned, sequenced and analyzed. Firstly, the evolutionary rate was calculated separately in the 3 codon positions. In all LTNPs, the 3rd codon mutation rate was equal or even lower than that observed at the 1st and 2nd positions (p = 0.016), thus suggesting strong ongoing positive selection. A Bayesian approach and a maximum-likelihood (ML) method were used to estimate the rate of virus evolution within each subject and to detect positively selected sites respectively. A great number of N-linked glycosylation sites under positive selection were identified in both NP and LTNP subjects. Viral sequences from 4 of the 5 LTNPs showed extensive positive selective pressure on the CD4-binding site (CD4bs). In addition, localized pressure in the area of the IgG-b12 epitope, a broad neutralizing human monoclonal antibody targeting the CD4bs, was documented in one LTNP subject, using a graphic colour grade 3-dimensional visualization. Overall, the data shown here documenting high selective pressure on the HIV-1 CD4bs of a group of LTNP subjects offers important insights for planning novel strategies for the immune control of HIV-1 infection.

SAX-HPLC and HSQC NMR Spectroscopy: Orthogonal Methods for Characterizing Heparin Batches Composition.

Heparin is a complex mixture of heterogeneous sulfated polysaccharidic chains. Its physico-chemical characterization is based on the contribution of several methods, but advantages of the use of complementary techniques have not been fully investigated yet. Strong-Anion-Exchange HPLC after enzymatic digestion and quantitative bidimensional 1H-13C NMR (HSQC) are the most used methods for the determination of heparin structure, providing the composition of its building blocks. The SAX-HPLC method is based on a complete enzymatic digestion of the sample with a mixture of heparinases I, II and III, followed by the separation of the resulting di- and oligo-saccharides by liquid chromatography. The NMR-HSQC analysis is performed on the intact sample and provides the percentage of mono- and di-saccharides by integration of diagnostic peaks. Since, for both methods, accuracy cannot be proved with the standard procedures, it is interesting to compare these techniques, highlighting their capabilities and drawbacks. In the present work, more than 30 batches of porcine mucosa heparin, from 8 manufacturers, have been analyzed with the two methods, and the corresponding results are discussed, based on similarities and differences of the outcomes. The critical comparison of both common and complementary information from the two methods can be used to identify which structural features are best evaluated by each method, and to verify from the concordance of the results the accuracy of the two methods, providing a powerful tool for the regular characterization of single, commercial preparations of Heparin.

1D and 2D-HSQC NMR: Two Methods to Distinguish and Characterize Heparin From Different Animal and Tissue Sources.

The US Food and Drug Administration has encouraged the reintroduction of bovine heparin drug product to the US market to mitigate the risks of heparin shortages and potential adulteration or contamination of the primary source which is porcine heparin. Here, a 1D-NMR method was applied to compare heparin sodium of bovine intestinal origin with that of bovine lung, porcine, or ovine intestinal origin. The results showed that a simple 1D test using NMR signal intensity ratios among diagnostic signals of the proton spectra uniquely identified the origin of heparin and concomitantly could be used to assure the correct sample labeling. However, a limitation of the use of only mono-dimensional spectra is that these spectra may not provide sufficiently detailed information on the composition of heparin batches to adequately determine the quality of this complex product. As an alternative, a higher resolution quantitative 2D-HSQC method was used to calculate the percentage of mono- and disaccharides, distinguish the origin of heparin and, simultaneously, assess the heparin composition. The 2D-HSQC method is proposed to provide sufficient information to evaluate the quality of industrial production process used to make the drug substance. Together, the 1D and 2D data produced by these measurements can be used to assure the identity and purity of this widely used drug.