Case Report: A child with NFKB1 haploinsufficiency explaining the linkage between immunodeficiency and short stature.

We report the case of a patient with common variable immunodeficiency (CVID) presenting with short stature and treated with recombinant human growth hormone (rhGH). Whole exome sequencing revealed a novel single-nucleotide duplication in the NFKB1 gene (c.904dup, p.Ser302fs), leading to a frameshift and thus causing NFKB1 haploinsufficiency. The variant was considered pathogenic and was later found in the patient's mother, also affected by CVID. This is the first reported case of a patient with CVID due to NFKB1 mutation presenting with short stature. We analyzed the interconnection between NFKB1 and GH - IGF-1 pathways and we hypothesized a common ground for both CVID and short stature in our patient.

Case Report: Zellweger Syndrome and Humoral Immunodeficiency: The Relevance of Newborn Screening for Primary Immunodeficiency.

Background: Zellweger syndrome (ZS) is a congenital autosomal recessive disease within the spectrum of peroxisome biogenesis disorders, characterized by the impairment of peroxisome assembly. The presence of peroxisome enzyme deficiencies leads to complex developmental sequelae, progressive disabilities, and multiorgan damage, due to intracellular accumulation of very-long-chain fatty acids (VLCFAs). Case Presentation: We report the case of an infant affected by ZS in which agammaglobulinemia, detected through neonatal screening of congenital immunodeficiencies, appeared as a peculiar trait standing out among all the other classical characteristics of the syndrome. The exome analysis through next-generation sequencing (NGS), which had previously confirmed the diagnostic suspicion of ZS, was repeated, but no mutations causative of inborn error of immunity (humoral defect) were detected. Conclusion: In this case, no genetic variants accountable for the abovementioned agammaglobulinemia were detected. Given that the scientific literature reports the involvement of peroxisomes in the activation of Nuclear Factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway, which is crucial for B-cell survival, with this work, we hypothesize the existence of a link between ZS and humoral immunodeficiencies. Further studies are required to confirm this hypothesis.

Bcgitis and vaccine-derived poliovirus infection in a patient with a novel deletion in RAG1 binding site.

A girl who developed severe BCGitis and vaccine-derived poliovirus infection was discovered to have a novel deletion of RAG1. A neonatal screening program for SCID would identify affected infants at birth, before live vaccines are administered.

Macrophage activation syndrome in a child affected by malaria: the choice of steroid.

Macrophage activation syndrome is a potentially fatal clinical syndrome caused by an excessive activation and proliferation of macrophages and T cells, leading to an exaggerated inflammatory reaction. It is well known that it can complicate the course of different conditions, especially autoimmune, lympho-proliferative, infectious diseases and drugs. Many infective pathogens can trigger the syndrome but the association with malaria has rarely been described, especially in children. We report a child with severe malaria complicated by MAS, in whom the clinical appearance of this syndrome could be considered as worsening of malaria itself. Furthermore, the use of steroids as first choice drugs in this complication, but arguable in malaria, has been highlighted. Clinicians should be aware of this syndrome when malaria does not respond to conventional therapy, since early diagnosis and prompt treatment may dramatically reduce the mortality associated with this condition.

Streptococcus pneumoniae: elusive mechanisms of the body's defense systems.

Streptococcus pneumoniae is one of the most important human pathogens. It represents the most frequent cause of pneumonia, meningitis, sinusitis and otitis. After the PCV7 vaccine introduction, a serotypic switch was noticed. This phenomenon led to the replacement of the seven serotypes contained in the vaccine with other less common ones, some of which are invasive or characterised by antibiotic-resistance. This replacement is only partially due to the vaccination. Many causes have been suggested to explain this effect: apearance of new serotypes, diffusion of minority serotypes and replacement of common serotypes due to natural secular trend. Pneumococcus has a promiscuous "sex life", characterized by homologous recombinations within the same species and also between different species. This fact can unlock the secret of how these pathogens can develop antibiotic or vaccine-resistance. The serotypic switch involves big loci that are responsible for capsular polysaccharide synthesis. The most important region of the genome involved in this process is near the gene tetM. The same mechanisms are also responsible for antibiotic resistance. In recent years the growth of penicillin, macrolides and clindamycine resistance has been noticed. It is also important to underline that multidrug-resistant bacteria isolation has increased. In conclusion, to obtain more information about bacteria composition and evolution, antibiotic-resistance and vaccine response, it is fundamental to improve the epidemiological surveillance of pneumococcal infections using modern molecular diagnostic techinques.

The immunity of upper airways.

A specialized immune system, called the nasopharynx-associated lymphoid tissue (NALT), is located on the mucosal surface of upper airways and is composed of both innate and specific immunity elements, closely related to each other and to the systemic defence, and spread over the nasopharynx, the nose and the middle ear. The NALT has a major role as regards to the infectious diseases of this area, which are the most common in children. It is also an interesting field of experimentation for new vaccine strategies in the future.

Chronic equine laminitis is characterised by loss of GLUT1, GLUT4 and ENaC positive laminar keratinocytes.

REASONS FOR PERFORMING STUDY: Equine laminitis is a multifactorial connective tissue disorder with major implications for the welfare of horses. There are few published studies on phenotypic markers for identification of equine laminar keratinocytes using immunohistochemical techniques. OBJECTIVES: To establish whether the epithelial sodium channel (ENaC) and the GLUT1 and GLUT4 facilitative glucose transporters may be used as phenotypic markers for identification of equine laminar keratinocytes using immunohistochemical techniques to monitor changes in the keratinocyte population in laminitis. METHODS: Histology and immunohistochemistry using polyclonal antibodies to the alpha subunit of ENaC (alphaENaC), GLUT1 and GLUT4 were used to compare the distribution of these proteins in normal and laminitic equine laminae. RESULTS: Immunohistochemistry with antibodies to alphaENaC, GLUT1 and GLUT4 confirmed the abundant expression of all 3 membrane proteins in healthy laminar keratinocytes. However, in laminitis, the Haematoxylin Van Gieson (HVG) technique revealed disordered laminar arrays and replacement with fibrous scar tissue. Immunostaining of laminitic samples confirmed the loss of alphaENaC, GLUT1 and GLUT4 positive keratinocytes. Other connective tissue cells did not stain positive for these proteins. CONCLUSIONS: This is the first report of alphaENaC and GLUT1/GLUT4 protein expression in equine laminar keratinocytes, which also confirms that the loss of laminar structure and function in chronic laminitis is accompanied by the loss of laminar keratinocytes. POTENTIAL RELEVANCE: alphaENaC, GLUT1 and GLUT4 may be used as phenotypic markers of metabolically active, differentiated equine laminar keratinocytes. Further in vitro studies are necessary to determine the effects of hypoxia, bacterial endotoxins, vasoactive amines, lactic acid and prostaglandins on the expression and activity of these plasma membrane keratinocyte markers.

Distribution and regulation of expression of serum- and glucocorticoid-induced kinase-1 in the rat kidney.

The serum- and glucocorticoid-induced kinase-1 (sgk1) increases the activity of a number of epithelial ion channels and transporters. The present study examines the distribution and subcellular localization of sgk1 protein in the rat kidney and the regulation of levels of expression induced by steroids. The results indicate that the kidney expresses predominantly the sgk1 isoform with a distribution restricted to the thick ascending limb of Henle, distal convoluted, connecting and cortical collecting tubules. Within cells, sgk1 strongly associates with the microsomal fraction of homogenates and it colocalizes with the Na+,K+-ATPase to the basolateral membrane. Analysis of the levels of expression of sgk1 by Western blotting and immunohistochemistry indicates constitutive high expression under basal conditions. Approximately half of the basal level is maintained by glucocorticoids whereas physiological fluctuations of aldosterone produce minor changes in sgk1 abundance in adrenal-intact animals. These results do not support the notion that physiological changes of aldosterone concentration turn the expression of sgk1 'on and off' in the mammalian kidney. Additionally, localization of sgk1 to the basolateral membrane indicates that the effects mediated by sgk1 do not require a direct interaction with the ion channels and transporters whose activity is modulated, since most of these proteins are located in the apical membrane of renal epithelial cells.

Anatomic study of the lymph nodes of the mesorectum.

PURPOSE: Lymph node involvement is the most important prognostic factor when staging patients with colorectal cancer. The probability of detecting metastasis grows with the number of nodes examined. However, the number of nodes found in surgical specimens varies substantially. We have therefore determined the number and distribution of lymph nodes in the mesorectum by cadaveric dissection. METHODS: Twenty formalin-fixed cadaveric pelvises were dissected (13 males). The search for lymph nodes was performed in a systematic way, from the division of the superior rectal artery following the smallest visible branches to the level of the anorectal ring. RESULTS: A total of 168 lymph nodes were found in 20 mesorectal blocks, with a mean (standard deviation) number per specimen of 8.4 (4.45). Lymph node size ranged from 2 to 10 mm. Distribution of lymph nodes in mesorectum was as follows: 120 nodes (71.4 percent) were found around the branches of the superior rectal artery proximal to the peritoneal reflection, and 48 nodes (28.6 percent) were found distal to the peritoneal reflection. Fourteen specimens (70 percent) had lymph nodes at the division of the superior rectal artery. CONCLUSIONS: The mean number of lymph nodes found in the mesorectum distal to the superior rectal artery division was 8.4. Most of these lymph nodes were proximal to the peritoneal reflection. The range found in the number of lymph nodes per case should be considered for use in the formulation of guidelines in anatomicopathologic studies of surgical specimens obtained after mesorectal excision.

Single-channel properties of recombinant acid-sensitive ion channels formed by the subunits ASIC2 and ASIC3 from dorsal root ganglion neurons expressed in Xenopus oocytes.

The acid-sensitive ion channels known as ASIC are gated by external protons. A set of these channels is expressed in dorsal root ganglion neurons where they may participate in the transduction of mechanical and nociceptive stimuli. Here, we have examined the single-channel properties of channels formed by the subunits ASIC2 and ASIC3 expressed in Xenopus oocytes using outside-out patches. The mean single-channel current-voltage relationship is linear with a slope conductance of 18 pS between -80 and -40 mV in 150 mM Na(+) outside and 150 mM K(+) inside the patch pipet. The selectivity for monovalent cations has the sequence Na(+) > Li(+) > K(+). Divalent cations such as Ca(2+) do not permeate, but instead block the channel when applied to the extracellular side. External protons increase the probability of channels being open to a maximum of 0.8 with an EC(50) of 16 +/- 4 microM and a Hill coefficient of 2.7 +/- 0.3, whereas the mean single-channel current amplitude is independent of external pH. Analysis of the kinetics of single channels indicates the presence of at least four modes of activity (Mod1 to Mod4) in addition to an inactivated state. Three of the modes exhibit distinct kinetics, and can be unambiguously identified on the basis of open probability (P(oMod1) = 0.5 +/- 0.05; P(oMod2) > 0.9 +/- 0.05; P(oMod3) < 0.1). Mode 4, which has a P(o) in the range of 0.5-0.8, may constitute a distinct mode or alternatively, it represents transitions between the other three modes of activity. Increasing [H(+)](o) increases the frequency of entering the modes with high P(o) (modes 1, 2, and 4) and the time the channel spends in the modes with high activity.

Heterologous expression of a mammalian epithelial sodium channel in yeast.

The alpha and beta subunits of the amiloride-sensitive rat epithelial sodium channel (alpha beta ENaC) were expressed in the yeast Saccharomyces cerevisiae. We used a combination of yeast strains, including a mutant in the secretory pathway (sec6), and Western blotting techniques, to show that alpha beta ENaC was synthesized and targeted through the secretory system to the plasma membrane. Yeasts expressing alpha beta ENaC were more sensitive to salt than the parent strain. In addition, amiloride, a specific blocker of ENaC, was found to suppress salt sensitivity in the yeast strain expressing alpha beta ENaC.

Structure and regulation of amiloride-sensitive sodium channels.

Amiloride-sensitive Na+ channels constitute a new class of proteins known as the ENaC-Deg family of ion channels. All members in this family share a common protein structure but differ in their ion selectivity, their affinity for the blocker amiloride, and in their gating mechanisms. These channels are expressed in many tissues of invertebrate and vertebrate organisms where they serve diverse functions varying from Na+ absorption across epithelia to being the receptors for neurotransmitters in the nervous system. Here, we review progress made during the last years in the characterization, regulation, and cloning of new amiloride-sensitive Na+ channels.

The second hydrophobic domain contributes to the kinetic properties of epithelial sodium channels.

The epithelial sodium channel (ENaC) is the prototype of a new class of ion channels known as the ENaC/Deg family. The hallmarks of ENaC are a high selectivity for Na(+), block by amiloride, small conductance, and slow kinetics that are voltage-independent. We have investigated the contribution of the second hydrophobic domain of each of the homologous subunits alpha, beta, and gamma to the kinetic properties of ENaC. Chimeric subunits were constructed between alpha and beta subunits (alpha-beta) and between gamma and beta subunits (gamma-beta). Chimeric and wild-type subunits were expressed in various combinations in Xenopus oocytes. Analysis of whole-cell and unitary currents made it possible to correlate functional properties with specific sequences in the subunits. Functional channels were generated without the second transmembrane domain from alpha subunits, indicating that it is not essential to form functional pores. The open probability and kinetics varied with the different channels and were influenced by the second hydrophobic domains. Amiloride affinity, Li(+)/Na(+) selectivity, and single channel conductance were also affected by this segment.

The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes.

The serum- and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloride-sensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of beta and gamma subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.

Inhibition of alphabeta epithelial sodium channels by external protons indicates that the second hydrophobic domain contains structural elements for closing the pore.

We have examined the effect of extracellular protons on the activity of epithelial sodium channels (ENaCs). We found that alphabeta channels, but not alphabetagamma or alphagamma channels, are inhibited by low extracellular pH. External protons induced short and long closed states that markedly decreased the open probability of alphabeta channels. External protons did not change the single-channel conductance or amiloride binding. Analysis of the proton-induced changes on the kinetics of single channels indicates that at least two protons sequentially bind to the extracellular domain at sites that are not in the ion pathway. Conformational changes induced by protonation of those sites are transmitted to the second hydrophobic domain (M2) of the subunits to induce closure of the pore. The results suggest that elements located in the carboxy-terminal half of M2 participate in the gating mechanism of ENaCs.

Sodium transport systems in human chondrocytes. II. Expression of ENaC, Na+/K+/2Cl- cotransporter and Na+/H+ exchangers in healthy and arthritic chondrocytes.

In this article, the second of two, we continue our studies of sodium-dependent transport systems in human cartilage from healthy individuals and with osteoarthritis (OA) and rheumatoid arthritis (RA). We demonstrate the presence of the epithelial sodium channel (ENaC), previously undescribed in chondrocytes. This system is composed of three subunits, alpha, beta and gamma. We have shown that the human chondrocytes express at least the alpha and the beta subunit of ENaC. The expression of these subunits is altered in arthritic chondrocytes. In RA samples the quantity of alpha and beta is significantly higher than in control samples. On the other hand, ENaC alpha and beta subunits are absent in the chondrocytes of OA cartilage. Human chondrocytes also possess three isoforms of the Na+/H+ exchanger (NHE), NHE1, NHE2 and NHE3. The NHE system is composed of a single protein and is believed to participate in intracellular pH regulation. Furthermore, our studies indicate that at least one isoform of the electroneutral Na+/K+/2Cl- cotransporter (NKCC) is present in human chondrocytes. There are no obvious variations in the relative expression of NHE isoforms or NKCC between healthy and arthritic cartilage. Our data suggests that chondrocytes from arthritic cartilage may adapt to changes in their environmental sodium concentration through variations in ENaC protein levels. ENaC is also likely to serve as a major sodium entry mechanism, a process that, along with cytoskeletal proteins, may be part of mechanotransduction in cartilage.

Evaluation of hematopoietic progenitors in hematopoietic progenitor cell transplants. CD34+ dose effect in marrow recovery. Retrospective analysis in 38 patients.

Our main goal was to evaluate the CD34+ dose in patients undergoing haemotopoietic stem celltransplantation and its results in terms of recovery of neutrophile and platelet counts, transfusion requirements, days of fever, antibiotic requirements and length of hospital stay. We studied 38 consecutive patients with haematological malignancies transplanted at our Department, from Feb. 96 through Sept. 98. The CD34+ cell quantification technique was standardized, using a modification of the ISAGHE 96 protocol. Patients were sorted into three groups according to the CD34+ count administered: a) between 3 and 5 x 10(6) cells/kg; b) between 5 and 10 x 10(6) cells/kg; c) > 10 x 10(6) CD34+ cells/kg. As a secondary end point, results were assessed according to the number of aphereses required to arrive at the target count of CD34+, separating those patients that required only 1 or 2 aphereses versus those requiring 3 or more. Finally, an analysis was made of the results of transplantation comparing the different sources of stem cells (PBSC versus PBSC + B.M.). The best results were obtained in the group with cells between 3 and 5 x 10(6) CD34+. No statistically significant advantages were found in the group with cells over 5. The supra-optimal dose of more 10 x 10(6) would yield no additional beneficial results, while they can imply a greater infusion of residual tumor cells. The number of aphereses had no impact on engraftment. Results obtained with PBSC transplants were better than those with BM+PBSC in terms of neutrophile and platelet recovery. The number of CD34+ cells remains the main element in stem cell transplantation to evaluate the haematopoietic recovery after engraftment. Minimum and optimum yields remain unclear. Centers should establish their own optimal dose based on local methodologies and outcomes, maximizing costs and benefits.

sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels.

The early phase of the stimulatory effect of aldosterone on sodium reabsorption in renal epithelia is thought to involve activation of apical sodium channels. However, the genes initiating this effect are unknown. We used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques to identify aldosterone-regulated immediate early genes in renal mineralocorticoid target cells. We report here that aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase, sgk (or serum- and glucocorticoid-regulated kinase), in its native target cells, i.e. in cortical collecting duct cells. The effect occurs within 30 min of the addition of aldosterone, is mediated through mineralocorticoid receptors, and does not require de novo protein synthesis. The full-length sequences of rabbit and mouse sgk cDNAs were determined. Both cDNAs show significant homology to rat and human sgk (88-94% at the nucleotide level, and 96-99% at the amino acid level). Coexpression of the mouse sgk in Xenopus oocytes with the three subunits of the epithelial Na+ channel results in a significantly enhanced Na+ current. These results suggest that sgk is an immediate early aldosterone-induced gene, and this protein kinase plays an important role in the early phase of aldosterone-stimulated Na+ transport.

Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes.

The epithelial sodium channel (ENaC) provides the rate-limiting step in the reabsorption of sodium by many epithelia. The number of channels at the cell surface is tightly regulated; most cells express only a few channels. We have examined the biosynthesis and cell surface expression of ENaC in Xenopus oocytes. The subunits of ENaC are readily synthesized in the endoplasmic reticulum, but most of them remain as immature proteins in pre-Golgi compartments, where they are degraded by the proteasomal pathway without apparent ubiquitination. Even when the three subunits, alpha, beta, and gamma, are expressed in the same cell, only a very small fraction of the total channel population leave the endoplasmic reticulum, acquire complex oligosaccharides, and reach the plasma membrane. Overexpression of subunits does not increase the number of channels in the plasma membrane but results in the appearance of cytoplasmic subunits in a form not membrane bound. The data indicate that maturation and assembly of the subunits are slow and inefficient processes, and constitute limiting steps for the expression of functional ENaC channels in the plasma membrane.

Subunit composition determines the single channel kinetics of the epithelial sodium channel.

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of alpha with either wild-type beta or gamma subunits and alpha with carboxy-terminal truncated beta (betaT) or gamma (gammaT) subunits in Xenopus laevis oocytes. alphabeta and alphabetaT channels (9.6 and 8.7 pS, respectively, with 150 mM Li+) were found to be constitutively open. Only upon inclusion of 1 microM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (Po) for alphabeta was 0.54 and for alphabetaT was 0.50. In comparison, alphagamma and alphagammaT channels exhibited different kinetics: alphagamma channels (6.7 pS in Li+) had either long open times with short closings, resulting in a high Po (0.78), or short openings with long closed times, resulting in a low Po (0. 16). The mean Po for all alphagamma channels was 0.48. alphagammaT (6.6 pS in Li+) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean Po of 0.6, similar to that of alphagamma. Inclusion of 0. 1 microM amiloride in the pipette solution reduced the mean open time of alphagammaT to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of alphabeta, alphabetaT (1 microM amiloride), and alphagammaT (0.1 microM amiloride) channels. alphabeta and alphabetaT had similar blocking and unblocking rate constants, whereas the unblocking rate constant for alphagammaT was 10-fold slower than alphabetaT. Our results indicate that subunit composition of ENaC is a main determinant of Po. In addition, channel kinetics and Po are not altered by carboxy-terminal deletion in the beta subunit, whereas a similar deletion in the gamma subunit affects channel kinetics but not Po.