Cone Beam-CT-Based Bone Volume Assessments of Alveolar Synthetic Bone Graft GlassBONE™ in Cleft Lip and Palate Patients: A Retrospective Study.

Background: Clefts of the lip and palate (CLP) are facial deformities that require multiple surgical procedures during childhood. One of these steps consists of filling the alveolar space with bone graft, traditionally removed from the iliac crest. However, this procedure could be invasive in children. Aim: Here, we aimed to evaluate the outcomes of GlassBONE™ graft, a bioactive glass used as a bone substitute, as an alternative to the deleterious autologous bone graft in children. Materials & methods: Retrospective monocentric study with 17 children aged 7.5 ± 2.2 yo [3.8-13.3 yo] carrying CLP. This technique has been established at La Timone Children hospital (Assistance Publique - Hôpitaux de Marseille) since 2011. Clinical (scar, graft rejection and periodontal status) and radiological (both panoramic radiographs and cone beam-CT) follow-up was conducted one year after the graft. The primary outcome was the reduction of the cleft volume, and secondary was the eruption of the adjacent tooth through the graft. Results: GlassBONE™ permitted a significant reduction in the cleft volume by 42.4 ± 27.7% [0.6-81.1%] (p < 0.0001), corresponding to a filling of 57.6 ± 27.7% of the alveolar cleft. GlassBONE™ is well tolerated, ensuring satifactory clinical results (improvement in both scar and periodontal coverage), as well as the physiological evolution of the germs through the biomaterial. GlassBONE™ appears particularly suitable for small volumes, and we were able to determine a minimum volume of approximtely 0.259 + / - 0.155 cc required for a successful bone fusion. Conclusion: The bioactive glass GlassBONE™ could be safely used in children with small CLP cases, providing satisfactory clinical and radiological results.

Willingness to vaccinate children against COVID-19 declined during the pandemic.

OBJECTIVES: To document the level of vaccine hesitancy in caregivers' of children younger than 12 years of age over the course of the pandemic in Pediatric Emergency Departments (ED). Study design Ongoing multicenter, cross-sectional survey of caregivers presenting to 19 pediatric EDs in the USA, Canada, Israel, and Switzerland during first months of the pandemic (phase1), when vaccines were approved for adults (phase2) and most recently when vaccines were approved for children (phase3). RESULTS: Willingness to vaccinate rate declined over the study period (59.7%, 56.1% and 52.1% in the three phases). Caregivers who are fully vaccinated, who have higher education, and those worried their child had COVID-19 upon arrival to the ED, were more likely to plan to vaccinate in all three phases. Mothers were less likely to vaccinate early in the pandemic, but this hesitancy attenuated in later phases. Older caregivers were more willing to vaccinate, and caregivers of older children were less likely to vaccinate their children in phase 3. During the last phase, willingness to vaccinate was lowest in those who had a primary care provider but did not rely on their advice for medical decisions (34%). Those with no primary care provider and those who do and rely on their medical advice, had similar rates of willingness to vaccinate (55.1% and 52.1%, respectively). CONCLUSIONS: COVID-19 vaccine hesitancy is widespread and growing over time, and public health measures should further try to leverage identified factors associated with hesitancy in order to enhance vaccination rates among children.

Factors associated with unvaccinated caregivers who plan to vaccinate their children.

Vaccine hesitancy is complex and a threat to global public health during the ongoing COVID-19 pandemic. Our objective was to determine factors associated with caregivers' willingness to vaccinate children despite not being immunized themselves against COVID-19. The International COVID-19 Parental Attitude Study (COVIPAS), a multinational cohort study, recruited caregivers of children 0-18 years old in 21 Emergency Departments (EDs) in USA, Canada, Israel, and Switzerland during November-December 2021. Of a total of 4536 caregivers who completed the survey, 882 (19.4%) were unvaccinated, and 62 (7.0%) of the unvaccinated planned to vaccinate their children. Unvaccinated caregivers with children that had their childhood vaccines up-to-date (OR 3.03 (1.36, 8.09), p = 0.01), and those very worried their child has COVID-19 in the ED (OR 3.11 (1.44, 6.34), p < 0.01) were much more likely to plan to immunize their children. Primary care providers and public health agencies should not assume that unvaccinated parents will not vaccinate their children. Determining child's vaccination status and parental level of concern about COVID-19 may help identify caregivers who are open to give their children the vaccine.

Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts.

Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3' splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.

Computational molecular phenotyping of retinal sheet transplants to rats with retinal degeneration.

Retinal progenitor sheet transplants have been shown to extend neuronal processes into a degenerating host retina and to restore visual responses in the brain. The aim of this study was to identify cells involved in transplant signals to retinal degenerate hosts using computational molecular phenotyping (CMP). S334ter line 3 rats received fetal retinal sheet transplants at the age of 24-40 days. Donor tissues were incubated with slow-releasing microspheres containing brain-derived neurotrophic factor or glial cell-derived neurotrophic factor. Up to 265 days after surgery, eyes of selected rats were vibratome-sectioned through the transplant area (some slices stained for donor marker human placental alkaline phosphatase), dehydrated and embedded in Eponate, sectioned into serial ultrathin datasets and probed for rhodopsin, cone opsin, CRALBP (cellular retinaldehyde binding protein), l-glutamate, l-glutamine, glutathione, glycine, taurine, γ-aminobutyric acid (GABA) and DAPI (4',6-diamidino-2-phenylindole). In large transplant areas, photoreceptor outer segments in contact with host retinal pigment epithelium revealed rod and cone opsin immunoreactivity whereas no such staining was found in the degenerate host retina. Transplant photoreceptor layers contained high taurine levels. Glutamate levels in the transplants were higher than in the host retina whereas GABA levels were similar. The transplant inner nuclear layer showed some loss of neurons, but amacrine cells and horizontal cells were not reduced. In many areas, glial hypertrophy between the host and transplant was absent and host and transplant neuropil appeared to intermingle. CMP data indicate that horizontal cells and both glycinergic and GABAergic amacrine cells are involved in a novel circuit between transplant and host, generating alternative signal pathways between transplant and degenerating host retina.

Retinal degeneration in two lines of transgenic S334ter rats.

Aim of this study was to examine synaptic connectivity changes in the retina and the location and rate of apoptosis in transgenic S334ter line-3 and line-5 rats with photoreceptor degeneration. Heterozygous S334ter-line-3 and line-5 at P11-13, P30, P60, P90 and several control non-dystrophic rats (Long Evans and Sprague-Dawley) at P60, were studied anatomically by immunohistochemistry for various cell and synaptic markers, and by PNA and TUNEL label.- S334ter line-3 exhibited the fastest rate of degeneration with an early loss of photoreceptors, with 1-2 layers remaining at P30, and only cones left at P60. Line-5 had 4-5 layers left at P30, and very few rods left at P60-90. In both lines, horizontal cell processes (including dendrites and axon) were diminished at P11-13, showing gaps in the outer plexiform layer (OPL) at P60, and at P90, almost no terminal tips could be seen. Bipolar cells showed a retraction of their dendrites forming clusters along the OPL. Synaptic terminals of A-II amacrine cells in the IPL lost most of their parvalbumin-immunoreactivity. The apoptosis rate was different in both lines. Line-3 rats showed many photoreceptors affected at P11, occupying the innermost part of the outer nuclear layer. Line-5 showed a lower number of apoptotic cells within the same location at P13. In summary, the S334ter line-3 rat has a faster progression of degeneration than line-5. The horizontal and bipolar terminals are already affected at P11-P13 in both models. Apoptosis is related to the mutated rhodopsin transgene; the first photoreceptor cells affected are those close to the OPL.

Visual restoration and transplant connectivity in degenerate rats implanted with retinal progenitor sheets.

The aim of this study was to determine whether retinal progenitor layer transplants form synaptic connections with the host and restore vision. Donor retinal sheets, isolated from embryonic day 19 rat fetuses expressing human placental alkaline phosphatase (hPAP), were transplanted to the subretinal space of 18 S334ter-3 rats with fast retinal degeneration at the age of 0.8-1.3 months. Recipients were killed at the age of 1.6-11.8 months. Frozen sections were analysed by confocal immunohistochemistry for the donor cell label hPAP and synaptic markers. Vibratome slices were stained for hPAP, and processed for electron microscopy. Visual responses were recorded by electrophysiology from the superior colliculus (SC) in 12 rats at the age of 5.3-11.8 months. All recorded transplanted rats had restored or preserved visual responses in the SC corresponding to the transplant location in the retina, with thresholds between -2.8 and -3.4 log cd/m(2). No such responses were found in age-matched S334ter-3 rats without transplants, or in those with sham surgery. Donor cells and processes were identified in the host by light and electron microscopy. Transplant processes penetrated the inner host retina in spite of occasional glial barriers between transplant and host. Labeled neuronal processes were found in the host inner plexiform layer, and formed apparent synapses with unlabeled cells, presumably of host origin. In conclusion, synaptic connections between graft and host cells, together with visual responses from corresponding locations in the brain, support the hypothesis that functional connections develop following transplantation of retinal layers into rodent models of retinal degeneration.

PCA and clustering reveal alternate mtDNA phylogeny of N and M clades.

Phylogenetic trees based on mtDNA polymorphisms are often used to infer the history of recent human migrations. However, there is no consensus on which method to use. Most methods make strong assumptions which may bias the choice of polymorphisms and result in computational complexity which limits the analysis to a few samples/polymorphisms. For example, parsimony minimizes the number of mutations, which biases the results to minimizing homoplasy events. Such biases may miss the global structure of the polymorphisms altogether, with the risk of identifying a "common" polymorphism as ancient without an internal check on whether it either is homoplasic or is identified as ancient because of sampling bias (from oversampling the population with the polymorphism). A signature of this problem is that different methods applied to the same data or the same method applied to different datasets results in different tree topologies. When the results of such analyses are combined, the consensus trees have a low internal branch consensus. We determine human mtDNA phylogeny from 1737 complete sequences using a new, direct method based on principal component analysis (PCA) and unsupervised consensus ensemble clustering. PCA identifies polymorphisms representing robust variations in the data and consensus ensemble clustering creates stable haplogroup clusters. The tree is obtained from the bifurcating network obtained when the data are split into k = 2,3,4,...,kmax clusters, with equal sampling from each haplogroup. Our method assumes only that the data can be clustered into groups based on mutations, is fast, is stable to sample perturbation, uses all significant polymorphisms in the data, works for arbitrary sample sizes, and avoids sample choice and haplogroup size bias. The internal branches of our tree have a 90% consensus accuracy. In conclusion, our tree recreates the standard phylogeny of the N, M, L0/L1, L2, and L3 clades, confirming the African origin of modern humans and showing that the M and N clades arose in almost coincident migrations. However, the N clade haplogroups split along an East-West geographic divide, with a "European R clade" containing the haplogroups H, V, H/V, J, T, and U and a "Eurasian N subclade" including haplogroups B, R5, F, A, N9, I, W, and X. The haplogroup pairs (N9a, N9b) and (M7a, M7b) within N and M are placed in nonnearest locations in agreement with their expected large TMRCA from studies of their migrations into Japan. For comparison, we also construct consensus maximum likelihood, parsimony, neighbor joining, and UPGMA-based trees using the same polymorphisms and show that these methods give consistent results only for the clade tree. For recent branches, the consensus accuracy for these methods is in the range of 1-20%. From a comparison of our haplogroups to two chimp and one bonobo sequences, and assuming a chimp-human coalescent time of 5 million years before present, we find a human mtDNA TMRCA of 206,000 +/- 14,000 years before present.

Antigen-specific tolerance of human alpha1-antitrypsin induced by helper-dependent adenovirus.

As efficient and less toxic virus-derived gene therapy vectors are developed, a pressing problem is to avoid immune response to the therapeutic gene product. Secreted therapeutic proteins potentially represent a special problem, as they are readily available to professional antigen-presenting cells throughout the body. Some studies suggest that immunity to serum proteins can be avoided in some mouse strains by using tissue-specific promoters. Here we show that expression of human alpha1-antitrypsin (AAT) was nonimmunogenic in the immune-responsive strain C3H/HeJ, when expressed from helper-dependent (HD) vectors using ubiquitous as well as tissue-specific promoters. Coadministration of less immunogenic HD vectors with an immunogenic first-generation vector failed to immunize, suggesting immune suppression rather than immune stealth. Indeed, mice primed with HD vectors were tolerant to immune challenge with hAAT emulsified in complete Freund's adjuvant. Such animals developed high-titer antibodies to coemulsified human serum albumin, showing that tolerance was antigen specific. AAT-specific T cell responses were depressed in tolerized animals, suggesting that tolerance affects both T and B cells. These results are consistent with models of high-dose tolerance of B cells and certain other suppressive mechanisms, and suggest that a high level of expression from HD vectors can be sufficient to induce specific immune tolerance to serum proteins.

Pathology of damaging electrical stimulation in the retina.

The goal of this study was to examine the characteristics of electrically induced retinal damage. A retinal prosthesis must be both effective and safe, but most research related to electrical stimulation of the retina has involved measures of efficacy (for example, stimulus threshold), while relatively little research has investigated the safety of electrical stimulation. In this study, a single platinum microelectrode was inserted into the vitreous cavity of normally-sighted adult Long Evans pigmented rats. In one group of animals, no contact was made between the electrode and the retina and current pulses of 0.05 (n=3) and 0.2 (n=6) microC/phase were applied. In a second group, visible contact (slight dimpling of the retina) was made between the electrode and the retina and current pulses of 0.09 (n=4) microC/phase were applied. In both cases, stimulus pulses (biphasic, cathodic first, 1 ms/phase) were applied for 1 h at 100 Hz. Also, control experiments were run with no electrical stimulation with retina contact (n=4) and with no retinal contact (n=3). After stimulation, the animal was survived for 2 weeks with ocular photography and electroretinography (ERG) to document changes. During the follow-up period, retinal changes were observed only when the electrode contacted the retina, with or without electrical stimulation. No difference was noted in ERG amplitude or latency comparing the test eye to the stimulated eye. Histological analysis was performed after sacrifice at 2 weeks. A semi-quantitative method for grading 18 features of retina/RPE/choroidal appearance was established and integer grades applied to both test and control eyes. Using this method and comparing the most severely affected area (highest grade), significant differences (p<0.05) were noted between experiments with retinal contact and without retinal contact in all features except inner nuclear layer thickness. No difference was noted within a group based on the intensity of electrical stimulus applied. The size of the affected area was significantly larger with both retinal contact and electrical stimulation compared to with retinal contact alone. We conclude that mechanical pressure alone and mechanical pressure with excessive electrical stimulation causes damage to the retina but that electrical stimulation coupled with mechanical pressure increases the area of the damage.

Heightened expression of the cytotoxicity receptor NKG2D correlates with acute and chronic nephropathy after kidney transplantation.

The activating cytotoxicity receptor NKG2D binds to stress-regulated molecules encoded by the major histocompatibility complex class I chain-related (MIC) and UL-16-binding protein (ULBP)/retinoic acid early transcript (RAET) gene family. To assess whether acute allograft rejection leads to an induction of these inducible ligands and their receptor NKG2D, we examined the mRNA profiles in kidney transplant biopsies. Expression levels were correlated with the incidence of acute rejection (aRx) episodes and chronic allograft nephropathy (CAN) proven by histology. Whereas MICA, ULBP1/3 and RAET1-E did not display heightened gene expression, elevated levels of NKG2D mRNA could be associated with aRx (p < 0.001). Immunohistology of kidney biopsies diagnosed with aRx revealed NKG2D+ cells in tubulointerstitial areas positive for CD8+ cells. Most importantly, elevated levels of NKG2D mRNA were associated with restricted long-term graft function assessed by the glomerular filtration rate at 6, 12 and 18 months posttransplantation. Induced NKG2D mRNA expression was still observable in biopsies diagnosed with CAN (p < 0.001), demonstrating a higher sensitivity and specificity compared to CD3, granzyme B and granulysin mRNA measurement. Significant elevated levels of NKG2D mRNA could be further detected in urine sediment prior to aRx, suggesting this receptor as a new candidate marker for the diagnosis of acute and chronic allograft rejection.

Helper-dependent adenoviral gene therapy mediates long-term correction of the clotting defect in the canine hemophilia A model.

BACKGROUND: Adenoviral vector-mediated gene therapy might have potential for long-term correction of the monogenic disease hemophilia A. OBJECTIVE: In this study, we tested the efficacy of administering a helper-dependent adenoviral vector (HDV) designed for maximal liver-restricted canine factor VIII (cFVIII) expression on three out-bred hemophilia A dogs. METHODS: Three FVIII-deficient animals from the University of North Carolina colony were injected with 1 x 10(12) (Dog A), and 3 x 10(12) (Dog B and C) vp kg(-1) helper-dependent adenoviral vector, and we performed systematic analysis of toxicity, persistence of therapeutic gene expression, and molecular analysis of gene transfer. RESULTS: We observed acute dose-dependent elevation in liver enzymes and thrombocytopenia after injection, although both were transient and resolved within 2 weeks. The whole blood clotting time (WBCT), plasma FVIII concentration, FVIII activity, and activated partial thromboplastin time in all animals improved significantly after treatment, and two animals receiving a higher dose reached near normal WBCT with low-level FVIII activity until terminal sacrifice at 3 months, and 2 years. Importantly, the treated dogs suffered no bleeding events after injection. Moreover, we observed persistent vector-specific DNA and RNA in liver tissue collected from one high-dose animal at days 18 and 79, and could not detect the formation of inhibitory antibodies. CONCLUSION: Although vector-associated toxicity remains an obstacle, a single injection of HDV led to long-term transgene expression and vector persistence in two FVIII-deficient animals with conversion of their severe phenotype to a moderate one.

Hyperbranched polymers as drug carriers: microencapsulation and release kinetics.

The aim of this work was to study the feasibility of hyperbranched polymers as drug carriers by employing different microparticle formation methods and the influence of loading methods on release kinetics. Commercially available hyperbranched polyester (Perstorp) and three polyesteramides (DSM) were loaded with the pharmaceutical acetaminophen. The gas antisolvent precipitation (GAS), the coacervation, and the particles from gas saturated solutions (PGSS) are among conventional processes that were used to prepare microparticles of drug-loaded hyperbranched polyesters for the first time. For preparing solid dispersions of drug-loaded hyperbranched polyesteramides the solvent method was applied. Infrared (IR) and differential thermal analysis (DTA) studies suggest that acetaminophen is partly dissolved in the polymer matrix and partly crystallized outside the polymer matrix. For acetaminophen-loaded polyesters prepared by the GAS method, the presence of free drugs is predominant when compared to microparticles prepared by the coacervation method. This event disappears for microparticles prepared by the PGSS method. Moreover, the release of drug from drug-loaded Bol-GAS is biphasic, where the initial burst (48%), indicating the presence of unincorporated drugs, is followed by a slow-release phase, suggesting the diffusion of drug through polymer matrices. The release of drugs from drug-loaded Bol-PGSS do not show this behavior since the drug is better dissolved or dispersed in polymer matrices. In the case of drug-loaded polyesteramides, coevaporates prepared from 3 hyperbranched structures (H1690, H1200, and H1500) using the solvent method result in different release kinetics. The hydrophobic characteristic of hyperbranched polyesteramide H1500 shows the biphasic release kinetic whereas the drug released from hydrophilic matrices H1690 and H1200 exhibits fast release comparable to that of pure drug.

Early post-transplant urinary IP-10 expression after kidney transplantation is predictive of short- and long-term graft function.

The early identification of renal transplant recipients at enhanced risk of developing acute and subclinical rejection would allow individualized adjustment of immunosuppression before functional graft injury occurs and would exclude these patients from drug-weaning studies. Protein and reverse transcriptase-polymerase chain reaction-based analyses of candidate markers in urine open the opportunity to closely monitor kidney-transplanted patients non-invasively. The chemokine interferon-inducible protein 10 (IP-10; CXCL10) might be an interesting candidate to uncover ongoing immune processes within the graft. Urine samples from kidney-transplanted recipients were retrospectively analyzed for IP-10 mRNA and protein expression. IP-10 levels were correlated with the incidence of acute rejection episodes proven by histology and long-term graft function assessed by the glomerular filtration rate 6 months post transplantation. IP-10 expression in urine identified patients with ongoing acute rejection episodes several days before a biopsy was indicated by rising serum creatinine levels. Most importantly, elevated levels of urinary IP-10 protein within the first four postoperative weeks were predictive of graft function at 6 months even in the absence of acute rejection. These data reveal a correlation between elevated IP-10 expression in urine at early time points post-transplantation and intragraft immune activation that leads to acute rejection and compromised long-term graft function.

Restoration of visual responses following transplantation of intact retinal sheets in rd mice.

PURPOSE: To correlate the functional outcomes with histologic findings following transplantation of fetal retinal sheets in rd mice, and to investigate the mechanisms of visual function restoration. METHODS: Twenty-one postnatal day 31-38 rd/rd (C3H/HeJ) mice were transplanted in one eye with retinal sheets (1.0 x 0.4 mm) obtained from embryonic day (E) 17 enhanced-green-fluorescent protein (eGFP) mice. Five mice underwent sham surgery without insertion of tissue. Four to five weeks after transplantation, visual responses to a light flash were recorded across the superior colliculus (SC) in seven eyes of seven transplanted mice that had clear corneas and lenses, and in all five sham surgery mice. Following the SC recording, the eyes were enucleated and processed for immunohistochemistry and examined using confocal microscopy. RESULTS: In three out of the seven eyes (43%), positive responses were recorded in the SC in an area topographically corresponding to the placement of the transplant in the host retina. No responses were recorded in the untreated eyes of 5-week-old and 9-week-old rd/rd mice, and in the 9-week-old sham surgery mice. In contrast, visual responses were recorded over the entire SC in normal eyes. The response onset latencies of the 3 transplanted mice with responses were similar to those of normal control mice. The organization of the graft did not appear to correlate as expected with the electrophysiology results, as eyes with well-organized, laminated grafts showed no response whereas the three light-responsive eyes had rosetted or disorganized grafts. All three light-responsive eyes demonstrated much higher levels of recoverin immunoreactivity in the host retina overlying the graft compared with untreated age-matched rd/rd mice. CONCLUSION: Restoration of the SC visual response does not appear to depend on a well-organized transplant in the rd mouse. Increased recoverin-staining in the host retina in light-responsive animals suggested that host cone rescue was the likely mechanism of vision restoration in this transplant model.

Alternative culture conditions for isolation and expansion of retinal progenitor cells.

PURPOSE: To investigate different in vitro model systems for retinal progenitor cell (RPC) isolation and expansion. METHODS: RPCs were isolated from embryonic day (E) 17 Long Evans rat retinas. Three different culture media: (1) modified serum free defined media (2) serum-containing media and (3) embryonic stem cell (ES)-conditioned media were used for RPC isolation and long term expansion. Expression of various cellular markers and cell morphologies were compared among the three culture systems at different passages by immunostaining and confocal microscopy. RESULTS: All three culture systems could maintain RPCs as nestin-positive cells (78-87%) after long-term in vitro expansion. However, RPCs appeared to proliferate faster in the serum-free culture system. The ES-conditioned media provided the best RPC survival. Cells appeared smaller at early passages compared with later passages. This morphology change occurred at P9-P10 in the serum-free medium, and at P5-P6 in the other two culture systems. CONCLUSIONS: The serum-free medium may be superior for preventing RPC differentiation during expansion.

High density oligonucleotide array analysis of interferon- alpha2a sensitivity and transcriptional response in melanoma cells.

Interferon alpha (IFN-alpha) represents an adjuvant therapy of proven effectiveness in increasing disease-free interval and survival in subgroups of melanoma patients. Since high doses of cytokine are required, the treatment is often accompanied by toxic side effects. Furthermore, naturally occurring insensitivity to IFN-alpha may hamper its therapeutic efficacy. Clinical, molecular or immunological markers enabling the selection of potential responders have not been identified so far. To explore the molecular basis of IFN-alpha responsiveness, we analysed the expression pattern of about 7000 genes in IFN-alpha sensitive and resistant cell lines and we compared the transcription profiles of cells cultured in the presence or absence of the cytokine using high-density oligonucleotide arrays. Melanoma cell lines were screened for their sensitivity to proliferation inhibition and HLA class I induction upon IFN-alpha treatment by standard 3H-thymidine incorporation and flow-cytometry. The study of 4 sensitive and 2 resistant cell lines allowed the identification of 4 genes (RCC1, IFI16, hox2 and h19) preferentially transcribed in sensitive cells and 2 (SHB and PKC-zeta) preferentially expressed in resistant cells. IFN-alpha stimulation resulted in the expression of a panel of 19 known inducible genes in sensitive but not in resistant cells. Moreover a group of 30 novel IFN-alpha inducible genes was identified. These data may provide a useful basis to develop diagnostic tools to select potential IFN-alpha responders eligible for treatment, while avoiding unnecessary toxicity to non-responders. Furthermore, by extending the knowledge of the polymorphic effects of IFN-alpha on gene expression, they offer novel clues to the study of its pleiotropic toxicity.

Retinal transplants restore visually evoked responses in rats with photoreceptor degeneration.

PURPOSE: To assess whether transplantation of intact sheets of fetal retina with retinal pigment epithelium (RPE) into a retina with photoreceptor degeneration restores visually evoked responses. METHODS: Sheets of fetal retina with RPE were transplanted into the subretinal space of Royal College of Surgeons (RCS) rats at 37 to 69 days of age. Sixty-three days to 10 months after transplantation, multiunit visual responses were recorded in the superior colliculus (SC) of transplanted rats, age-matched untransplanted rats, and rats with sham surgery. RESULTS: In 19 of 29 RCS rats with transplants, visually evoked responses were recorded from and restricted to a small area of the SC that corresponds topographically to the portion of the retina in which the transplant was placed. Outside of this area, no visual responses were evoked. Visually evoked responses were never recorded in age-matched, nontransplanted RCS rats. Visually evoked responses were recorded in 6 of 13 RCS rats with sham surgery, but these responses were significantly different from responses in rats with transplants. CONCLUSIONS: These results demonstrate that this transplantation technique restores visually evoked responses in the brain. Although the underlying mechanism is unknown, we propose that the central visual response results from increased synaptic efficacy within the host retina. If it can be established that functional connections between the transplant and the host retina produce the effect, then it would indicate that the technique could be explored as a therapeutic strategy in some diseases of retinal degeneration.

The osmolyte xylitol reduces the salt concentration of airway surface liquid and may enhance bacterial killing.

The thin layer of airway surface liquid (ASL) contains antimicrobial substances that kill the small numbers of bacteria that are constantly being deposited in the lungs. An increase in ASL salt concentration inhibits the activity of airway antimicrobial factors and may partially explain the pathogenesis of cystic fibrosis (CF). We tested the hypothesis that an osmolyte with a low transepithelial permeability may lower the ASL salt concentration, thereby enhancing innate immunity. We found that the five-carbon sugar xylitol has a low transepithelial permeability, is poorly metabolized by several bacteria, and can lower the ASL salt concentration in both CF and non-CF airway epithelia in vitro. Furthermore, in a double-blind, randomized, crossover study, xylitol sprayed for 4 days into each nostril of normal volunteers significantly decreased the number of nasal coagulase-negative Staphylococcus compared with saline control. Xylitol may be of value in decreasing ASL salt concentration and enhancing the innate antimicrobial defense at the airway surface.

Endoscopic detection of dysplasia in patients with Barrett's esophagus using light-scattering spectroscopy.

BACKGROUND & AIMS: We conducted a study to assess the potential of light-scattering spectroscopy (LSS), which can measure epithelial nuclear enlargement and crowding, for in situ detection of dysplasia in patients with Barrett's esophagus. METHODS: Consecutive patients with suspected Barrett's esophagus underwent endoscopy and systematic biopsy. Before biopsy, each site was sampled by LSS using a fiberoptic probe. Diffusely reflected white light was spectrally analyzed to obtain the size distribution of cell nuclei in the mucosal layer, from which the percentage of enlarged nuclei and the degree of crowding were determined. Dysplasia was assigned if more than 30% of the nuclei exceeded 10 microm and the histologic findings compared with those of 4 pathologists blinded to the light-scattering assessment. The data were then retrospectively analyzed to further explore the diagnostic potential of LSS. RESULTS: Seventy-six sites from 13 patients were sampled. All abnormal sites and a random sample of nondysplastic sites were reviewed by the pathologists. The average diagnoses were 4 sites from 4 different patients as high-grade dysplasia (HGD), 8 sites from 5 different patients as low-grade dysplasia (LGD), 12 as indefinite for dysplasia, and 52 as nondysplastic Barrett's. The sensitivity and specificity of LSS for detecting dysplasia (either LGD or HGD) were 90% and 90%, respectively, with all HGD and 87% of LGD sites correctly classified. Decision algorithms using both nuclear enlargement and crowding further improved diagnostic accuracy, and accurately classified samples into the 4 histologic categories. CONCLUSIONS: LSS can reliably detect LGD and HGD in patients with Barrett's esophagus.