Posttransplantation late complications increase over time for patients with SCID: A Primary Immune Deficiency Treatment Consortium (PIDTC) landmark study.

BACKGROUND: The Primary Immune Deficiency Treatment Consortium (PIDTC) enrolled children in the United States and Canada onto a retrospective multicenter natural history study of hematopoietic cell transplantation (HCT). OBJECTIVE: We investigated outcomes of HCT for severe combined immunodeficiency (SCID). METHODS: We evaluated the chronic and late effects (CLE) after HCT for SCID in 399 patients transplanted from 1982 to 2012 at 32 PIDTC centers. Eligibility criteria included survival to at least 2 years after HCT without need for subsequent cellular therapy. CLE were defined as either conditions present at any time before 2 years from HCT that remained unresolved (chronic), or new conditions that developed beyond 2 years after HCT (late). RESULTS: The cumulative incidence of CLE was 25% in those alive at 2 years, increasing to 41% at 15 years after HCT. CLE were most prevalent in the neurologic (9%), neurodevelopmental (8%), and dental (8%) categories. Chemotherapy-based conditioning was associated with decreased-height z score at 2 to 5 years after HCT (P < .001), and with endocrine (P < .001) and dental (P = .05) CLE. CD4 count of ≤500 cells/µL and/or continued need for immunoglobulin replacement therapy >2 years after transplantation were associated with lower-height z scores. Continued survival from 2 to 15 years after HCT was 90%. The presence of any CLE was associated with increased risk of late death (hazard ratio, 7.21; 95% confidence interval, 2.71-19.18; P < .001). CONCLUSION: Late morbidity after HCT for SCID was substantial, with an adverse impact on overall survival. This study provides evidence for development of survivorship guidelines based on disease characteristics and treatment exposure for patients after HCT for SCID.

Measuring the effect of newborn screening on survival after haematopoietic cell transplantation for severe combined immunodeficiency: a 36-year longitudinal study from the Primary Immune Deficiency Treatment Consortium.

BACKGROUND: Severe combined immunodeficiency (SCID) is fatal unless durable adaptive immunity is established, most commonly through allogeneic haematopoietic cell transplantation (HCT). The Primary Immune Deficiency Treatment Consortium (PIDTC) explored factors affecting the survival of individuals with SCID over almost four decades, focusing on the effects of population-based newborn screening for SCID that was initiated in 2008 and expanded during 2010-18. METHODS: We analysed transplantation-related data from children with SCID treated at 34 PIDTC sites in the USA and Canada, using the calendar time intervals 1982-89, 1990-99, 2000-09, and 2010-18. Categorical variables were compared by χ2 test and continuous outcomes by the Kruskal-Wallis test. Overall survival was estimated by the Kaplan-Meier method. A multivariable analysis using Cox proportional hazards regression models examined risk factors for HCT outcomes, including the variables of time interval of HCT, infection status and age at HCT, trigger for diagnosis, SCID type and genotype, race and ethnicity of the patient, non-HLA-matched sibling donor type, graft type, GVHD prophylaxis, and conditioning intensity. FINDINGS: For 902 children with confirmed SCID, 5-year overall survival remained unchanged at 72%-73% for 28 years until 2010-18, when it increased to 87% (95% CI 82·1-90·6; n=268; p=0·0005). For children identified as having SCID by newborn screening since 2010, 5-year overall survival was 92·5% (95% CI 85·8-96·1), better than that of children identified by clinical illness or family history in the same interval (79·9% [69·5-87·0] and 85·4% [71·8-92·8], respectively [p=0·043]). Multivariable analysis demonstrated that the factors of active infection (hazard ratio [HR] 2·41, 95% CI 1·56-3·72; p<0·0001), age 3·5 months or older at HCT (2·12, 1·38-3·24; p=0·001), Black or African-American race (2·33, 1·56-3·46; p<0·0001), and certain SCID genotypes to be associated with lower overall survival during all time intervals. Moreover, after adjusting for several factors in this multivariable analysis, HCT after 2010 no longer conveyed a survival advantage over earlier time intervals studied (HR 0·73, 95% CI 0·43-1·26; p=0·097). This indicated that younger age and freedom from infections at HCT, both directly driven by newborn screening, were the main drivers for recent improvement in overall survival. INTERPRETATION: Population-based newborn screening has facilitated the identification of infants with SCID early in life, in turn leading to prompt HCT while avoiding infections. Public health programmes worldwide can benefit from this definitive demonstration of the value of newborn screening for SCID. FUNDING: National Institute of Allergy and Infectious Diseases, Office of Rare Diseases Research, and National Center for Advancing Translational Sciences.

TREC Screening for WHIM Syndrome.

PURPOSE: T cell receptor excision circle (TREC) quantification is a recent addition to newborn screening (NBS) programs and is intended to identify infants with severe combined immunodeficiencies (SCID). However, other primary immunodeficiency diseases (PID) have also been identified as the result of TREC screening. We recently reported a newborn with a low TREC level on day 1 of life who was diagnosed with WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome, a non-SCID primary immunodeficiency caused by mutations in the chemokine receptor CXCR4. METHODS: We have now retrospectively reviewed the birth and clinical histories of all known WHIM infants born after the implementation of NBS for SCID. RESULTS: We identified six infants with confirmed WHIM syndrome who also had TREC quantification on NBS. Three of the six WHIM infants had low TREC levels on NBS. All six patients were lymphopenic but only one infant had a T cell count below 1,500 cells/µL. The most common clinical manifestation was viral bronchiolitis requiring hospitalization. One infant died of complications related to Tetralogy of Fallot, a known WHIM phenotype. CONCLUSION: The results suggest that WHIM syndrome should be considered in the differential diagnosis of newborns with low NBS TREC levels. TRIAL REGISTRATION: Not applicable.

Tonic inhibition of chronic pain by neuropeptide Y.

Dramatically up-regulated in the dorsal horn of the mammalian spinal cord following inflammation or nerve injury, neuropeptide Y (NPY) is poised to regulate the transmission of sensory signals. We found that doxycycline-induced conditional in vivo (Npy(tet/tet)) knockdown of NPY produced rapid, reversible, and repeatable increases in the intensity and duration of tactile and thermal hypersensitivity. Remarkably, when allowed to resolve for several weeks, behavioral hypersensitivity could be dramatically reinstated with NPY knockdown or intrathecal administration of Y1 or Y2 receptor antagonists. In addition, Y2 antagonism increased dorsal horn expression of Fos and phosphorylated form of extracellular signal-related kinase. Taken together, these data establish spinal NPY receptor systems as an endogenous braking mechanism that exerts a tonic, long-lasting, broad-spectrum inhibitory control of spinal nociceptive transmission, thus impeding the transition from acute to chronic pain. NPY and its receptors appear to be part of a mechanism whereby mammals naturally recover from the hyperalgesia associated with inflammation or nerve injury.

Emx2 stimulates odorant receptor gene expression.

The mechanisms selecting a single odorant receptor (OR) gene for expression in each olfactory sensory neuron (OSN) establish an OR expression pattern critical for odor discrimination. These mechanisms are largely unknown, but putative OR promoters contain homeodomain-like sites, implicating homeobox transcription factors such as Emx2. At embryonic day 18.5, expression of 49-76% of ORs was decreased in mice lacking Emx2, depending on the metric used. The decreases were due to fewer OSNs expressing each OR. Affected ORs showed changes that were disproportionately greater than the 42% reduction in mature neurons and similar decreases in unrelated olfactory neuron-enriched messenger RNAs in Emx2(-/-) mice. Both Class I and Class II ORs decreased, as did ORs expressed in both the dorsal and ventral regions of the epithelium. Conversely, 7% of Class II ORs tested were expressed more frequently, suggesting that some ORs are independent of Emx2. Emx2 helps stimulate transcription for many OR genes, which we hypothesize is through direct action at OR promoters, but Emx2 appears to have no significant role in regulating other aspects of OR gene expression, including the zonal patterns, OR gene cluster selection mechanisms, and singularity of OR gene choice.

Tissue expression patterns identify mouse cilia genes.

In mammals, cilia are critical for development, sensation, cell signaling, sperm motility, and fluid movement. Defects in cilia are causes of several congenital syndromes, providing additional reasons to identify cilia-related genes. We hypothesized that mRNAs selectively abundant in tissues rich in highly ciliated cells encode cilia proteins. Selective abundance in olfactory epithelium, testes, vomeronasal organ, trachea, and lung proved to be an expression pattern uniquely effective in identifying documented cilia-related genes. Known and suspected cilia-related genes were statistically overrepresented among the 99 genes identified, but the majority encoded proteins of unknown function, thereby predicting new cilia-related proteins. Evidence of expression in a highly ciliated cell, the olfactory sensory neuron, exists for 73 of the genes. In situ hybridization for 17 mRNAs confirmed expression of all 17 in olfactory sensory neurons. Most were also detected in vomeronasal sensory neurons and in neighboring tissues rich in ciliated cells such as respiratory epithelium. Immunoreactivity for one of the proteins identified, Spa17, colocalized with acetylated tubulin in the cilia layer of the olfactory epithelium. In contrast, the ciliary rootlet protein, Crocc, was located in discrete structures whose position was consistent with the dendritic knobs of the olfactory sensory neurons. A compilation of >2,000 mouse genes predicted to encode cilia-related proteins revealed a strong correlation (R = 0.99) between the number of studies predicting a gene's involvement in cilia and documented evidence of such involvement, a fact that simplifies the selection of genes for further study of the physiology of cilia.

Mouse olfactory sensory neurons express 10,000 genes.

Olfactory epithelial cells from olfactory marker protein-green fluorescent protein (OMP-GFP) mice were separated by fluorescence-activated cell sorting into a GFP+ sample enriched in mature olfactory sensory neurons (OSNs) and a GFP- sample enriched in all other cells. GeneChip expression profiling of these samples provided a predictive measure of expression in OSNs. Validation tests comparing the ratio of GFP+/GFP- signal intensity against expression patterns from in situ hybridization for 189 mRNAs proved statistically significant and provided probabilities of expression in OSNs scaled according to the signal intensity ratios. These probabilities predict that, among 11,596 mRNAs detected in the GFP+ sample, more than 10,000 are expressed in OSNs. Transcripts and overrepresented categories of mRNAs detected in the GFP+ sample agreed with known properties of OSNs and predict additional properties. For example, ciliogenesis and spermatogenesis were overrepresented, consistent with similarities between OSN cilia and sperm flagella. Chromatin assembly mRNAs were expressed throughout the OSN cell lineage, consistent with the hypothesis that chromatin remodeling plays a role in OSN differentiation. We detected numerous signaling proteins and receptors, such as 30 nonchemosensory G-protein-coupled receptors, including the presynaptic glutamate receptor mGlur4 and the Wnt receptor Fzd3. The largest group of mRNAs, however, was the hundreds of transcriptional regulators that presumably determine the OSN phenotype. The absence of OMP protein in OMP-GFP mice had no detectable effect on mRNA abundance. Within limits prescribed by the nature of microarray data and the in situ hybridization validation, these data should be useful in directing further experiments on OSN function.

Transcriptional changes during neuronal death and replacement in the olfactory epithelium.

The olfactory epithelium has the unusual ability to replace its neurons. We forced replacement of mouse olfactory sensory neurons by bulbectomy. Microarray, bioinformatics, and in situ hybridization techniques detected a rapid shift in favor of pro-apoptotic proteins, a progressive immune response by macrophages and dendritic cells, and identified or predicted 439 mRNAs enriched in olfactory sensory neurons, including gene silencing factors and sperm flagellar proteins. Transcripts encoding cell cycle regulators, axonogenesis proteins, and transcription factors and signaling proteins that promote proliferation and differentiation were increased at 5--7 days after bulbectomy and were expressed by basal progenitor cells or immature neurons. The transcription factors included Nhlh 1, Hes 6, Lmyc 1, c-Myc, Mxd 4, Id 1, Nmyc 1, Cited 2, c-Myb, Mybl 1, Tead 2, Dp 1, Gata 2, Lmo 1, and Sox1 1. The data reveal significant similarities with embryonic neurogenesis and make several mechanistic predictions, including the roles of the transcription factors in the olfactory sensory neuron lineage.

Differentially expressed transcripts from phenotypically identified olfactory sensory neurons.

In comparing purified mouse olfactory sensory neurons (OSNs) with neighboring cells, we identified 54 differentially expressed transcripts. One-third of the transcripts encode proteins with no known function, but the others have functions that correlate with challenges faced by OSNs. The OSNs expressed a diversity of signaling protein genes, including stomatin (Epb7.2), S100A5, Ddit3, Sirt2, CD81, Sdc2, Omp, and Ptpla. The elaboration of dendrites, cilia, and axons that places OSNs in contact with diverse cell types and signals presumably also requires large investments in cytoskeletal-associated proteins, lipid biosynthesis, and energy production. Several of the genes encode proteins that participate in these biological processes, including ATP5g3, Ndufa9, Sqrdl, Mdh1, Got1, beta-2 tubulin, Capza1, Bin3, Tom1, Acl6, and similar to O-MACS. Three transcripts had restricted expression patterns. Similar to O-MACS and Gstm2 had zonally restricted expression patterns in OSNs and sustentacular cells but not in Bowman's glands, suggesting that zonality can be differentially regulated by cell type. The mosaic expression pattern of S100A5 in approximately 70% of OSNs predicts that it is coexpressed with a subset of odorant receptors. We captured four abundant transcripts, Cyp2a4, similar to Cyp2g1, Gstm2, and Cbr2, that encode xenobiotic metabolizing enzymes expressed by sustentacular cells or Bowman's glands, reinforcing the interpretation that clearance of xenobiotic compounds is a major function of these cells. Within the olfactory epithelium, Cbr2 is a new anatomical marker for sustentacular cells. We also discovered that Reg3g is a marker for respiratory epithelium.

Transcriptional changes during neuronal death and replacement in the olfactory epithelium.

The olfactory epithelium has the unusual ability to replace its neurons.We forced replacement of mouse olfactory sensory neurons by bulbectomy. Microarray, bioinformatics, and in situ hybridization techniques detected a rapid shift in favor of pro-apoptotic proteins, a progressive immune response by macrophages and dendritic cells, and identified or predicted 439 mRNAs enriched in olfactory sensory neurons, including gene silencing factors and sperm flagellar proteins. Transcripts encoding cell cycle regulators, axonogenesis proteins, and transcription factors and signaling proteins that promote proliferation and differentiation were increased at 5-7 days after bulbectomy and were expressed by basal progenitor cells or immature neurons. The transcription factors included Nhlhl, Hes6, Lmycl, c-Myc, Mxd4, Idl,Nmycl, Cited2, c-Myb, Mybll, Tead2, Dpl, Gata2, Lmol, and Soxll. The data reveal significant similarities with embryonic neurogenesis and make several mechanistic predictions, including the roles of the transcription factors in the olfactory sensory neuron lineage.

Investigation of the role of macrophages on the cytotoxicity of doxorubicin and doxorubicin-loaded nanoparticles on M5076 cells in vitro.

Doxorubicin-loaded PACA nanoparticles have been shown to be more efficient than free drug in mice bearing hepatic metastasis of the M5076 tumour. Due to the high phagocytic activity of Küpffer cells in the liver, it may be that these cells played a role of drug reservoir after nanoparticle phagocytosis. Therefore, the objective of this study was to assess the role of macrophages in mediating the cytotoxicity of doxorubicin-loaded nanoparticles on M5076 cells. The growth inhibition of tumour cells was evaluated in two ways: firstly, the cells were incubated in a coculture system consisting of special wells with two compartments separated by a porous membrane. M5076 cells were seeded into the lower compartment and the macrophages J774.A1 were introduced into the upper part. The macrophages were activated or not by IFN-gamma. The drug preparations were added only in the macrophage insert. Secondly, growth inhibition was also assessed in the conventional way, i.e. in direct contact with the tumour cells to serve as a reference. After direct contact, free doxorubicin (Dox) and doxorubicin-loaded nanoparticles (NP-Dox) had the same efficacy against M5076 cell growth. The coculture experiments led to a 5-fold increase in the IC(50) for both Dox and NP-Dox. The activation of macrophages by IFN-gamma in coculture significantly decreased the IC(50) values. In conclusion, after phagocytosis of doxorubicin-loaded nanoparticles, J774.A1 cells were able to release active drug, allowing it to exert its cytotoxicity against M5076 cells. Drug efficacy was potentiated by the activation of macrophages releasing cytotoxic factors such as NO, which resulted in increased tumour cell death. Thereby, the coculture system permitted us to investigate the macrophage-mediated cytotoxicity of colloidal carriers loaded with an anticancer drug, which is of great interest when further i.v. administration is envisaged.

Reversion of multidrug resistance by co-encapsulation of doxorubicin and cyclosporin A in polyalkylcyanoacrylate nanoparticles.

Individual and combined polyalkylcyanoacrylate nanoparticle formulation of cyclosporin A and doxorubicin were prepared and evaluated in an attempt to show improved growth inhibition efficacy in a resistant cell culture line. The drug loaded nanoparticles were prepared using the well established emulsion polymerization process without using any modification for the hydrophilic doxorubicin drug whereas the incorporation of cyclosporin A needed to wait a moment after the polymerization reaction started. This was necessary to avoid cyclosporin A precipitation and polymer aggregation. Cyclosporin A release from the nanoparticles was rapid probably because the drug was adsorbed onto the nanoparticles surface rather than embedded into the polymeric core. Doxorubicin displayed also a burst effect but with a slower second phase probably related with the nanoparticles bioerosion rate owing to its entrapment in the polymeric network. Finally, it was shown in resistant cell culture experiments that the association of both cyclosporin A and doxorubicin within a single nanoparticle formulation elicited the most effective growth rate inhibition as compared to other combinations of both drugs while using a lower amount of polymer compared to separated nanoparticle formulations. This result was probably due to the synergistic effect achieved by combining the chemo-sensitizing compound cyclosporin A, with an effective cytotoxic drug like doxorubicin.

Ability of doxorubicin-loaded nanoparticles to overcome multidrug resistance of tumor cells after their capture by macrophages.

PURPOSE: Investigation of the ability of doxorubicin-loaded nanoparticles (NP/Dox) to overcome multidrug resistance (MDR) when they have first been taken up by macrophages. METHODS: The growth inhibition of P388 sensitive (P388) and resistant (P388/ADR) tumor cells was evaluated in a coculture system consisting of wells with two compartments. The tumor cells were seeded into the lower compartment, the macrophages were introduced into the upper part in which the drug preparations were also added. RESULTS: Doxorubicin exerted lower cytotoxicity on tumor cells in coculture compared with direct contact. In P388/ADR, NP/Dox cytotoxicity was far higher than that of free doxorubicin (Dox). Three different formulations of cyclosporin A (either free (CyA), loaded to nanoparticles (NP/CyA) or in a combined formulation with doxorubicin (NP/Dox-CyA)), were added to modulate doxorubicin efficacy. The addition of cyclosporin A to Dox increased drug cytotoxicity. Both CyA added to NP/Dox and NP/Dox-CyA were able to bypass drug resistance. CONCLUSIONS: Despite the barrier role of macrophages, NP/Dox remained far more cytotoxic than Dox against P388/ADR. Both NP/Dox + CyA and NP/Dox-CyA allowed to overcome MDR, but the last one should present greater advantage in vivo by confining both drugs in the same compartment, hence reducing the adverse effects.

[The role of transplanted vein graft in arterial reconstruction]. / Il ruolo dell'innesto con vena traslocata nella ricostruzione arteriosa.

The use of the non-reversed saphenous vein has not been properly evaluated yet, for what concerns the features of length and application. In the reported series this technique has been employed for the reconstruction of arterial segments in various anatomic sites. The mean follow-up was 10 months. Between November 1987 and September 1993 at the Department of Vascular Surgery of Pietra Ligure and Imperia 39 arterial grafts with the use of non-reversed autologous saphenous vein were performed, for the treatment of arteriosclerotic, traumatic or aneurysmatic diseases. On the basis of the outcomes (70 to 100% patency of the grafts found at the follow-up), pro and cons of this technique is weighed up and some guidelines about the procedure of choice (among the non-reversed, in situ or reversed saphenous vein) are settled, keeping in mind the different variations who must influence and direct the surgeons' choice (venous diameter, features of the site to revascularize etc.).

Repair of acute Achilles tendon ruptures.

Acute Achilles tendon injuries are discussed in terms of various etiologies, pathomechanics, and treatment options. Surgical management has evolved to promote the athlete's early return to sport utilizing a new suture technique and aggressive rehabilitation protocol. The specific training and recovery modalities have significantly reduced the morbidity of these injuries by restoring strength, range of motion, and preventing disuse atrophy.

Achilles tendon disorders.

In conclusion, the sports medicine specialist is able to use a spectrum of diagnostic, surgical, and rehabilitation techniques to identify etiologic factors and to choose optimal treatment regimens for patients with Achilles tendinitis or traumatic rupture. Correction of pathomechanic factors, anatomic restoration, and aggressive postoperative rehabilitation allows an early return to sport without significant loss of strength or mobility.

Diagnosis and treatment of iliopsoas abscess in spinal cord injury patients.

Six patients with spinal cord injury and iliopsoas abscess and other complicating conditions were evaluated with computed tomography (CT), conventional radiography, magnetic resonance imaging (MRI), and radionuclide scans. CT identified the presence of psoas abscess and revealed the depth, extent, and relationship of deep pressure ulcers to deep structures. CT-guided aspiration of the abscess cavities was performed in three patients, with placement of drainage catheters. Concurrent treatment with appropriate antibiotics, followed by staged myocutaneous flap coverage resulted successful outcomes in all patients. A high index of suspicion aids in the early diagnosis of psoas abscess in the SCI patient, as interpretations of physical examination are obscured by the lack of localizing findings. We believe that CT is the diagnostic and therapeutic modality of choice in the management of these complex conditions in the SCI patient, because of its superior ability to detect pathologic changes in the pelvic region and for decreasing the morbidity of the treatment by avoiding open surgery in these often suboptimal surgical candidates.

Magnetic resonance imaging of the hindfoot.

This article demonstrates normal anatomy of the foot and ankle as visualized with magnetic resonance imaging (MRI) in the sagittal, axial, and coronal planes. Additionally, selected cases chosen from our experience with more than 100 clinical scans are shown to highlight the primary areas in which we have found MRI to be clinically useful: bone marrow abnormalities, especially osteomyelitis and osteonecrosis, soft tissue injuries and masses, and cases in which metallic fixators make CT evaluation problematic.

Excimer ablation of human intervertebral disc at 308 nanometers.

Excimer laser energy, which has been shown to photoablate tissue at a precisely controllable rate with minimal thermal damage, was applied to human intervertebral disc in an effort to develop a technique for percutaneous discectomy. Cadaveric samples of human disc were used. Excimer laser energy was produced by a XeCl, magnetically switched, long-pulse laser working at 308 nm, 20 Hz. Annulus tissue of approximately 1 mm thickness was placed in contact with the output tip of a 400 microns core diameter quartz fiber, and measurements of ablation rate were made at different radiant exposures. Ablation rates were found to vary linearly with radiant exposure, from 0.7 micron/pulse at 10 mJ/mm2 to 11.0 microns/pulse at 55 mJ/mm2, with a correlation coefficient of 0.984. Threshold radiant exposure, calculated by extrapolation, was found to be about 7 mJ/mm2. Histologic analysis showed a minimum of thermal damage in these specimens, and when ablated with modification to maintain constant fiber-tissue contact, thermal injury was nearly absent, as compared to samples ablated with Nd:YAG through a contact probe. Thermographic analysis, performed using the AGA 782 Digital Thermography system, showed increasing temperature with increasing radiant exposure, with a maximum temperature of 47.2 degrees C at 55 mJ/mm2. In that precise tissue ablation was demonstrated with minimal generated heat, and excimer energy at 308 nm is transmissible through fiber optics, excimer holds great promise for the development of a percutaneous discectomy technique.