C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques

Background. Detection of C4d along peritubular capillaries (PTC) in renal allograft biopsies is an independent prognostic marker of poor long-term graft survival. It is typically associated with circulating donor-specific antibodies. Since only little information is available on the best technique to stain C4d, we compared the two methods most often used for detecting C4d in renal allograft specimens. Methods. We investigated the expression of C4d along PTC in 64 renal allograft biopsies using a monoclonal antibody (Quidel) and immunofluorescence for frozen (F-IF) and a polyclonal antibody (Biomedica) and immunohistochemistry for formalin-fixed and paraffin-embedded (P-IHC) tissue samples. We compared the staining extent (diffuse, focal, minimal, no staining) in frozen and paraffin sections and evaluated the intra- and inter-observer concordance rates using kappa statistics. In addition, we determined the inter-observer concordance in 240 paraffin-embedded biopsies of a multi-centre study. Results. The inter- and intra-investigator concordance rate (κ = 0.9) of analysing the C4d expression by F-IF was excellent. In contrast, the detection of C4d by P-IHC demonstrated a substantially lower prevalence and extent of C4d expression with a lower intra- and inter-observer concordance rate (κ = 0.3). Only 69% of diffuse and 13% of focal C4d-expressing cases were in line classified by F-IF and P-IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by P-IHC than by F-IF. The inter-observer concordance rate in paraffin of the 64 renal biopsies and the multi-centre study was good, but not perfect (κ = 0.57 or 0.67). Conclusions. C4d staining determined on frozen tissue samples using F-IF with a monoclonal antibody appears to be better suited for diagnostic as well as research purposes. Future studies should correlate C4d staining patterns with circulating donor-specific antibodie

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease

Background. Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease. Methods. Frozen sections of renal biopsies were stained with monoclonal antibodies to TLR-2, -4 and -9. Results. Up-regulation of the three TLRs studied was seen, although the extent was modest. TLR-2- and -4-positive cells belonged to the population of infiltrating inflammatory cells; only in the case of TLR-9 were intrinsic glomerular cells positive in polyoma virus infection and haemolytic uraemic syndrome (HUS). Conclusions. Evidence for the involvement of the three TLRs tested in a variety of human renal diseases was found. These findings add to our understanding of the role of the innate immune system in kidney diseas

BK virus large T and VP-1 expression in infected human renal allografts

Objective. We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology

BK virus large T and VP-1 expression in infected human renal allografts

Objective. We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology. Methods. Frozen sections from three patients with renal allografts (two biopsies, one explant) with PVAN were analysed by indirect immunofluorescence using BKV specific anti-polyoma large T-antigen and anti-VP-1 antibodies, as well as anti-p53, anti-Ki67, anti-caspase-3, anti-bcl2 and anti-cytokeratin 22 antibodies. Nuclear morphology and size were estimated by DNA Hoechst staining. Results. In infected tubular cells the early and late phases of infection could be distinguished according to expression of large T-antigen or VP-1. The early phase revealed almost normal nuclear proportions, whereas in later phases nuclear size increased about 2 to 3 fold. Expression of large T-antigen was strongly associated with accumulation of p53 in the nucleus, accompanied by the activation of the cell cycle associated cell protein Ki67. In contrast, expression of BKV VP1 correlated only weakly with p53. Virus dependent cell lysis was due to necrosis, since neither caspase 3 nor nuclear nor cytoskeleton changes indicated apoptosis. Conclusion. In our selected patients with PVAN a clear distinction between early and late phases was possible, according to the protein expression patterns of BKV markers. Striking nuclear enlargement is only present in the late phase of infection. In the inflammatory setting of PVAN, BKV dependent effects appear to be mediated by the inhibition of p53, resulting in the activation of the cell cycle. We assume that in PVAN similar BKV mechanisms are operative as in certain in vitro system

Alternativen zu Säuglingsnahrungen auf Kuhmilchproteinbasis

Die natürliche Ernährung eines gesunden Säuglings ist das Stillen. Ist Stillen oder die Fütterung von Muttermilch nicht möglich, kann Frauenmilch für die Ernährung des Säuglings erwogen werden, sofern diese aus einer qualifizierten Humanmilchbank stammt. Vom Kauf von Frauenmilch aus dem Internet wird wegen Risiken einer Infektionsübertragung und einer unzureichenden Milchqualität strikt abgeraten. Aufgrund der geringen Verfügbarkeit, der hohen Kosten sowie möglicher Nachteile der Ernährung mit Spendermilch im Vergleich zum Stillen sind industriell gefertigte Säuglingsnahrungen das Mittel der Wahl zur Ernährung des gesunden, reifgeborenen Säuglings, wenn Stillen nicht oder nur partiell möglich ist. Kuhmilchprotein ist die am häufigsten verwendete Eiweißkomponente. Die Nachfrage nach anderen auf Tiermilchen basierten sowie vegetarischen bzw. veganen Alternativen steigt. Im Folgenden werden verschiedene Alternativen bezüglich ihrer Eignung betrachtet. Säuglingsnahrungen auf Basis von Ziegenmilchprotein stellen für gesunde, reifgeborene Säuglinge eine zugelassene und geeignete Alternative zu kuhmilchproteinbasierten Säuglingsnahrungen dar. Für Säuglingsnahrungen aus anderen Tiermilchen (z. B. Kamel‑, Schaf‑, Pferde- oder Büffelmilch) sind keine belastbaren Daten zu Eignung und Sicherheit bekannt, und sie sind in der Europäischen Union nicht zugelassen. Säuglingsnahrungen auf der Grundlage von Sojaproteinisolaten sind in der Europäischen Union zugelassen. Sie werden für die allgemeine Verwendung im 1. Lebenshalbjahr durch die Ernährungskommission nicht empfohlen, insbesondere weil potenziell nachteilige Effekte von enthaltenen Isoflavonen nicht ausgeschlossen werden können. Ab der Geburt und in den ersten Lebensmonaten sollte die Gabe von Sojanahrungen auf Indikationen wie eine bestehende Galaktosämie, die sehr seltene kongenitale Laktoseintoleranz sowie bei familiärem Wunsch nach veganer Ernährungsweise und aus anderen weltanschaulichen Gründen begrenzt werden. Im 2. Lebenshalbjahr ist die Zufuhrmenge pro Kilogramm Körpergewicht deutlich niedriger, sodass das Risiko unerwünschter Wirkungen als wesentlich geringer eingeschätzt wird. Zu neuerdings angebotenen Nahrungen auf der Grundlage einer Mischung aus Soja- und Kuhmilchprotein sind keine Daten zur Prüfung von Sicherheit und Eignung bekannt, sodass hierzu keine Empfehlung ausgesprochen werden kann. Von der Verwendung von Säuglingsnahrung auf der Grundlage von hydrolysiertem Reisprotein wird auch aufgrund hoher berichteter Arsengehalte abgeraten. Auch von einer häuslichen Selbstherstellung von Säuglingsnahrungen wird aufgrund eines erhöhten Risikos für eine nichtbedarfsgerechte Nährstoffzufuhr und für Infektionen abgeraten. // The natural nutrition of a healthy infant is breastfeeding. If breastfeeding or feeding of breast milk is not possible, feeding of donor human milk may be considered. The safety of donor milk can only be ensured if it is provided by a qualified human milk bank. The use of informally shared donor milk or the use of human milk purchased through the internet is strongly discouraged because of the risk of transmitting infections and of insufficient milk quality. Due to low availability, high costs and concerns about poorer nutritional quality of donor milk compared to breastfeeding, industrially manufactured infant formula is the preferred alternative for feeding healthy full-term infants that cannot or cannot be fully breastfed. Milk-based infant formula is most frequently made from cow’s milk protein; however, there is an increasing popularity of vegetarian or vegan formulas and of formulas based on different animal milks other than cow’s milk. Goat’s milk-based infant formulas represent an approved and suitable alternative to cow’s milk for healthy, full-term infants. Reliable data on the suitability and safety are unavailable for formulas based on other animal milks (e.g., camel, horse, sheep or buffalo milk), and these are not approved for infant feeding in the European Union. Infant formulas based on soybean protein isolates are approved in the European Union. They are not generally recommended by the Nutrition Committee for infant feeding in the first half year of life because potentially harmful effects of isoflavones derived from soybeans cannot be excluded; however, soybean protein isolate-based formulas may be reservedly used after birth and in the first months of life for infants affected by galactosemia, the very rare hereditary lactose intolerance manifest at birth, a vegan family lifestyle or other familial convictions. In the second half year of life the intake amount per kilogram body weight is much less so that the risk of undesired effects is estimated to be much lower. For the recently provided nutrition based on a mixture of soybean and cow’s milk proteins, no data on testing of the safety and suitability are known, so that no recommendations can be made on this. The use of infant formula based on hydrolyzed rice protein is not recommended because of concerns about possible high arsenic contamination. The use of homemade infant formulas is discouraged due to the high risk of introducing infections and of possible nutritional imbalances of macronutrients and micronutrients

ESPGHAN/ESPEN/ESPR/CSPEN guidelines on pediatric parenteral nutrition

Background: Previous guidelines on Paediatric Parenteral Nutrition (PN) were published in 2010, by the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the European Society for Clinical Nutrition and Metabolism (ESPEN), supported by the European Society of Paediatric Research (ESPR) were published. The aim of the present paper was to provide up-to-date evidence for health professionals working with infants, children and adolescents receiving PN. Methods: The current document is a revision of the 2005 guidelines produced by the same 3 organizations (ESPEN, ESPGHAN, ESPR) together with the Chinese Society of Parenteral and Enteral Nutrition (CSPEN). Experts participating in the guideline updating process were all professionals with extensive experience in managing PN from a wide range of European countries, Israel and China. The guideline development process was coordinated by a guideline steering committee. Each chapter of the guideline was prepared by a separate author group. These author groups were responsible for screening titles and abstracts identified by a systematic literature search for inclusion, for conducting additional expert searches (including secondary sources such as other published valid guidelines), for evaluating the quality of studies included in the given chapter and assigning evidence levels to the literature. Based on the evidence level of included studies experts formulated and graded recommendations. A consensus conference was held in February 2015. All chapter manuscripts were revised following the recommendations of the consensus conference and then reviewed and edited by the project steering committee. Final consensus on each individual guideline and its individual recommendations was achieved and assessed by online voting. This process lasted until January 2018. Funding for the consensus conference (including travel expenses for participants) was provided by all participating societies. No other funding was received for the guideline updating process and participants received no payment. Support was provided by the Hungarian Cochrane organization. Results/conclusions: The present document provides guideline for the use of PN across the wide range of pediatric patients, ranging from extremely premature infants up to teenagers weighing up to and over 100 kg [1]. It covers their individual macro- and micronutrient needs [2], [3], [4], [5], [6], [7], [8], fluid requirements [9], venous access [10], organizational aspects [11], home parenteral nutrition [12], standardized vs. individualized PN [13], and last but not least a wide range of safety considerations for prevention and management of complications such central line associated bloodstream infections (CLABSI) [14]

Iron requirements of infants and toddlers

Iron deficiency (ID) is the most common micronutrient deficiency worldwide and young children are a special risk group because their rapid growth leads to high iron requirements. Risk factors associated with a higher prevalence of ID anemia (IDA) include low birth weight, high cow's-milk intake, low intake of iron-rich complementary foods, low socioeconomic status, and immigrant status. The aim of this position paper was to review the field and provide recommendations regarding iron requirements in infants and toddlers, including those of moderately or marginally low birth weight. There is no evidence that iron supplementation of pregnant women improves iron status in their offspring in a European setting. Delayed cord clamping reduces the risk of ID. There is insufficient evidence to support general iron supplementation of healthy European infants and toddlers of normal birth weight. Formula-fed infants up to 6 months of age should receive iron-fortified infant formula, with an iron content of 4 to 8 mg/L (0.6-1.2 mg(-1) · kg(-1) · day(-1)). Marginally low-birth-weight infants (2000-2500 g) should receive iron supplements of 1-2 mg(-1) · kg(-1) · day(-1). Follow-on formulas should be iron-fortified; however, there is not enough evidence to determine the optimal iron concentration in follow-on formula. From the age of 6 months, all infants and toddlers should receive iron-rich (complementary) foods, including meat products and/or iron-fortified foods. Unmodified cow's milk should not be fed as the main milk drink to infants before the age of 12 months and intake should be limited to <500 mL/day in toddlers. It is important to ensure that this dietary advice reaches high-risk groups such as socioeconomically disadvantaged families and immigrant families

C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques

BACKGROUND: Detection of C4d along peritubular capillaries (PTC) in renal allograft biopsies is an independent prognostic marker of poor long-term graft survival. It is typically associated with circulating donor-specific antibodies. Since only little information is available on the best technique to stain C4d, we compared the two methods most often used for detecting C4d in renal allograft specimens. METHODS: We investigated the expression of C4d along PTC in 64 renal allograft biopsies using a monoclonal antibody (Quidel) and immunofluorescence for frozen (F-IF) and a polyclonal antibody (Biomedica) and immunohistochemistry for formalin-fixed and paraffin-embedded (P-IHC) tissue samples. We compared the staining extent (diffuse, focal, minimal, no staining) in frozen and paraffin sections and evaluated the intra- and inter-observer concordance rates using kappa statistics. In addition, we determined the inter-observer concordance in 240 paraffin-embedded biopsies of a multi-centre study. RESULTS: The inter- and intra-investigator concordance rate (kappa = 0.9) of analysing the C4d expression by F-IF was excellent. In contrast, the detection of C4d by P-IHC demonstrated a substantially lower prevalence and extent of C4d expression with a lower intra- and inter-observer concordance rate (kappa = 0.3). Only 69% of diffuse and 13% of focal C4d-expressing cases were in line classified by F-IF and P-IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by P-IHC than by F-IF. The inter-observer concordance rate in paraffin of the 64 renal biopsies and the multi-centre study was good, but not perfect (kappa = 0.57 or 0.67). CONCLUSIONS: C4d staining determined on frozen tissue samples using F-IF with a monoclonal antibody appears to be better suited for diagnostic as well as research purposes. Future studies should correlate C4d staining patterns with circulating donor-specific antibodies