Induction of vaginal-resident HIV-specific CD8 T cells with mucosal prime-boost immunization.

Tissue-resident memory (TRM) CD8 T cells survey a range of non-lymphoid mucosal tissues where they rapidly mediate clearance of viral infections at the entry portals. Vaccines that establish CD8 TRM cells in the cervicovaginal mucosa hold promise for effective immunity against sexually transmitted HIV. We demonstrate that HIV-specific CD8 TRM cells can be established in the murine vaginal mucosa using a combined intranasal and intravaginal mucosal immunization with recombinant influenza-HIV vectors. Using in situ tetramer immunofluorescence microscopy, we found that this mucosally administered prime-boost immunization also resulted in the durable seeding of CD8 T cells in the frontline vaginal epithelial compartment as opposed to the vaginal submucosa. Upon cognate antigen recognition within the vaginal mucosa, these HIV-specific CD8 TRM cells rapidly initiated a tissue-wide state of immunity. The activation of HIV-specific CD8 TRM cells resulted in the upregulation of endothelial vessel addressin expression and substantial recruitment of both adaptive and innate immune cells in the vaginal mucosa. These findings suggest that the epithelial localization of HIV-specific CD8 TRM cell populations and their capacity to rapidly activate both arms of the immune system could significantly augment frontline defenses against vaginal HIV infection.

Downregulation of interleukin-18-mediated cell signaling and interferon gamma expression by the hepatitis B virus e antigen.

UNLABELLED: The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE: It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

Role of monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity.

Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. A widely used method to measure ADCC Abs is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. Antibody-dependent killing of a labelled target cell line by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+). Cells of this phenotype are assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We assessed the effector cells that mediate ADCC in this assay. Backgating analysis and phenotyping of CFSE-PKH26+ revealed that the RFADCC assay's readout mainly represents CD3-CD14+ monocytes taking up the PKH26 dye. This was confirmed for 53 HIV+plasma-purified IgG samples when co-cultured with PBMC from three separate healthy donors. Emergence of the CFSE-PKH26+ monocyte population was observed upon co-culture of targets with purified monocytes but not with purified NK cells. Image flow cytometry and microscopy showed a monocyte-specific interaction with target cells without typical morphological changes associated with phagocytosis, suggesting a monocyte-mediated ADCC process. We conclude that the RFADCC assay primarily reflects Ab-mediated monocyte function. Further studies on the immunological importance of HIV-specific monocyte-mediated ADCC are warranted.

Thoracoscopy in children: anaesthesiological implications and case reports.

AIM: Videoassisted thoracic surgical technique in children is being used with increasing frequency for an extensive variety of diagnostic and therapeutic procedures. The aim of the study was to assess respiratory, cardiocirculatory and body temperature changes in children undergoing thoracoscopy and to identify if the trend of such changes was modifiable by factors such as lung exclusion, length of the thoracoscopy and preoperative respiratory compromise. METHODS: A total of 50 patients (38 boys and 12 girls) undergoing general anaesthesia for diagnostic and therapeutic thoracoscopic procedures were analysed. The values of the monitored parameters were compared at 6 specific times: T1 - at the end of anaesthesia induction (considered the basal level); T2 - after lateral position; T3 - before pleural CO2 insufflation; T4 - 10 min after pleural CO2 insufflation; T5 - before pleural deflation; T6 - 10 min after pleural deflation. RESULTS: All patients tolerated the thoracoscopy well, without intraoperative complications. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were significantly lower, and end-tidal CO2 (PETCO(2) significantly higher during thoracoscopy. Body temperature (BT) had a statistically significant reduction during thoracoscopy and after pleural deflation. During one-lung ventilation the PETCO(2) increased compared to two-lung ventilation with intrapleural insufflation, while during two-lung ventilation with intrapleural insufflation SBP and DBP decreased compared to one-lung ventilation. The length of the thoracoscopy increased the PETCO(2) and reduced the BT. The preoperative respiratory compromise increased the PETCO(2). CONCLUSIONS: Although thoracoscopy in children brings about certain respiratory, cardiocirculatory and body temperature changes, it is nevertheless a safe and efficient surgical technique.

[Thoracoscopic treatment of oesophageal atresia]. / Correzione toracoscopica dell'atresia esofagea.

BACKGROUND: Advancements in minimally invasive surgery in newborns have allowed even the most complex neonatal procedures to be approached using these techniques. Other authors have demonstrated its efficacy in the treatment of the esophageal atresia with distal fistula. METHODS: We report our experience based on the thoracoscopic repair of esophageal atresia with distal fistula in two newborns. Birth weights were 2.800 g and 2.300 g respectively. The patients were intubated endotracheally and placed in a left prone position. Four trocars were inserted: the first one of 5 mm was positioned in the fifth intercostal space for the camera, the other two of 3 mm were positioned in the fourth intercostal space on the anterior and posterior axillary line respectively for the operative instruments. The last trocar of 3 mm was inserted in the third intercostal space on the anterior axillary line for the lung retractor. CO2 was insufflated at a pressure of 8 mm Hg and a flow of 0.5 L/min. The fistula was first isolated then ligated and cut with scissors. The proximal esophagus was opened and an anastomosis was made over a 6F or 8F nasogastric tube with interrupted 4-0 Vicryl sutures. A tube chest was placed through the lower trocar site with the tip near the anastomosis. RESULTS: These two procedures were free of neither intraoperative nor post-operative complications. Feeding by nasogastric tube was started after a mean of 4 days. Barium swallow made on day 7 demonstrated no leakage and no stenosis of the anastomosis. Total oral feeding was possible after 8 days. Mean hospitalization was 14 days. CONCLUSION: This initial report shows, as demonstrated by the experience since 1999 by other authors, that the thoracoscopic esophageal repair in the newborns is technically feasible and, thanks to a magnified vision, it allows to abtain a good isolation of the esophagus and of the tracheo-esophageal fistula respecting the anatomical structures. Moreover the advantages are in terms of exposure and esophageal length, avoiding the significant short and long-term morbidity associated with thoracotomy.

Enhanced cellular immunity in macaques following a novel peptide immunotherapy.

Advances in treating and preventing AIDS depend on understanding how human immunodeficiency virus (HIV) is eliminated in vivo and on the manipulation of effective immune responses to HIV. During the development of assays quantifying the elimination of fluorescent autologous cells coated with overlapping 15-mer simian immunodeficiency virus (SIV) or HIV-1 peptides, we made a remarkable observation: the reinfusion of macaque peripheral blood mononuclear cells, or even whole blood, pulsed with SIV and/or HIV peptides generated sharply enhanced SIV- and HIV-1-specific T-cell immunity. Strong, broad CD4+- and CD8+-T-cell responses could be enhanced simultaneously against peptide pools spanning 87% of all SIV- and HIV-1-expressed proteins-highly desirable characteristics of HIV-specific immunity. De novo hepatitis C virus-specific CD4+- and CD8+-T-cell responses were generated in macaques by the same method. This simple technique holds promise for the immunotherapy of HIV and other chronic viral infections.

Efficacy of DNA and fowlpox virus priming/boosting vaccines for simian/human immunodeficiency virus.

Further advances are required in understanding protection from AIDS by T-cell immunity. We analyzed a set of multigenic simian/human immunodeficiency virus (SHIV) DNA and fowlpox virus priming and boosting vaccines for immunogenicity and protective efficacy in outbred pigtail macaques. The number of vaccinations required, the effect of DNA vaccination alone, and the effect of cytokine (gamma interferon) coexpression by the fowlpox virus boost was also studied. A coordinated induction of high levels of broadly reactive CD4 and CD8 T-cell immune responses was induced by sequential DNA and fowlpox virus vaccination. The immunogenicity of regimens utilizing fowlpox virus coexpressing gamma interferon, a single DNA priming vaccination, or DNA vaccines alone was inferior. Significant control of a virulent SHIV challenge was observed despite a loss of SHIV-specific proliferating T cells. The outcome of challenge with virulent SHIV(mn229) correlated with vaccine immunogenicity except that DNA vaccination alone primed for protection almost as effectively as the DNA/fowlpox virus regimen despite negligible immunogenicity by standard assays. These studies suggest that priming of immunity with DNA and fowlpox virus vaccines could delay AIDS in humans.

[In-hospital pediatric cardiopulmonary resuscitation]. / Emergenze intraospedaliere pediatriche.

Between 0,7-3% of pediatric patients may require resuscitation during hospital stay. The physicians of the Pediatric Intensive Care Unit of the C.O.U. Anesthesia and Intensive Care-Baroncini developed a plan for the management of pediatric emergencies inside the Department of Pediatric Medical and Surgical Sciences. The plan consisted of: the drawing up of a PI 34-Procedure; the preparation and implementation of a training course for doctors and nurses; the purchase of 12 emergency-trolleys according to the Broselow Pediatric Resuscitation Measuring Tape and the implementation of a specific system for the emergency-call. Precise duty for anesthesiologists and intensivists is the emergency- planning and management, in order to diffuse the medical knowledge needed to assist patients requiring vital functions support. The management of the intra-hospital pediatric emergencies is strictly dependent on the training of the staff, based on a specific support algorithm, and specific equipment for the different ages.

Ovine interleukin-12: analysis of biologic function and species comparison.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced mainly by phagocytic and antigen-presenting cells (APC). The cDNA encoding the ovine IL-12 (OvIL-12) subunits, p40 and p35, were generated from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). The ovine genes encoded proteins that had the highest amino acid identity to caprine p40 (99% amino acid identity) and p35 (97% amino acid identity) and also displayed a high degree of identity with human p40 (84%) and p35 (79%) homologs. To ensure the equal expression of both subunits, we used the self-cleaving properties of the 2A oligopeptide from foot-and-mouth disease virus (FMDV) to express IL-12 as a single, long open reading frame (ORF) encoding p402Ap35. Using an in vitro transcription/translation system, we demonstrated that this 2A oligopeptide mediated cleavage of the p402Ap35 into p402A and p35, in a manner similar to the processing of the FMDV polypeptide. Moreover, when expressed in COSm6 cells, this self-processing polypeptide encoded a functional heterodimer, which elicited biologic activities associated with IL-12 in other species.

The expression and biologic effects of ovine interleukin-4 on T and B cell proliferation.

Using the reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA encoding ovine (Ov) interleukin-4 (OvIL-4) was generated from mitogen-stimulated peripheral blood mononuclear cells (PBMC). Two identical clones generated from separate RT-PCR reactions differed from a published OvIL-4 sequence, although they had a high degree of identity with the bovine and human homologs. We show by sequence analysis that the OvIL-4 cDNA retained the four alpha-helix structure and disulfide bonds identified in human IL-4 (HuIL-4). Moreover, the cDNA encoding OvIL-4 was expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Supernatants from insect cells infected with the recombinant virus secreted an additional protein with a relative molecular mass of 17,000. This protein was recognized by an anticervine IL-4 monoclonal antibody (mAb) in a Western blot and did not react with any proteins in supernatants from uninfected insect cells or cells infected with the wild-type AcMNPV. Supernatants from insect cells infected with the recombinant virus induced the proliferation of activated B cells in a dose-dependent manner and typically demonstrated 5 x 105 dilution U/ml of activity. However, OvIL-4 had no effect on the proliferation of resting T cells isolated from efferent lymph and actually inhibited the ability of a mitogen to stimulate these resting lymphocytes. In contrast, OvIL-4 induced the proliferation of mitogen-activated lymphoblast, demonstrating the complex role(s) OvIL-4 plays in the regulation of B and T cells.

Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep.

Vaccination of sheep with a plasmid bearing the full length gene for the tick antigen Bm86 either alone or co-administered with plasmid carrying the ovine genes for the cytokines, granulocyte and macrophage colony stimulating factor (GM-CSF) or interleukin (IL)-1beta induced a relatively low level of protection against subsequent tick infestation. This tick damage reached statistical significance only for the groups which were vaccinated with plasmid encoding for Bm86, co-administered with plasmid encoding for ovine GM-CSF. Antibody titres measured against Bm86 were also low in all groups injected with the Bm86 DNA vaccine. Antibody production and anti-tick effect were significantly less than that achieved by two vaccinations with recombinant Bm86 protein. In all cases only a low level of antigen-specific stimulation of peripheral blood lymphocytes was recorded, as measured either by the incorporation of tritiated thymidine or the release of IFN-gamma. Injection of DNA encoding for Bm86, either alone or with co-administered cytokine genes, did however prime for a strong subsequent antibody response following a single injection of recombinant Bm86 protein in adjuvant. Antibody production nevertheless appeared to be slightly less effective than following two vaccinations with recombinant protein. The persistence of antibody following vaccination was the same regardless of the method of primary sensitization. In all cases the half-life of the antibody response was approximately 40-50 days indicating that, in contrast to results reported in mice, DNA vaccination in sheep did not result in sustained antibody production.

Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in sheep.

A large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (DeltaPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting DeltaPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding DeltaPLD. While all groups of sheep vaccinated with DNA encoding DeltaPLD were afforded better protection against an experimental challenge with C. pseudotuberculosis than those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.

[Inhalation of foreign bodies]. / Inalazione di corpi estranei nelle vie aeree.

Accidental aspiration of a foreign body (FB) is an event which is reasonably frequent and dramatic in children and is still today one of the main causes of death due to accidents at home in children up to three-four years of age. The severity of the clinical picture varies according to the size, shape, type and site of arrest of the material aspirated and can be associated with both severe asphyxial forms and forms with insidious and vague symptoms which are difficult to diagnose correctly. A late diagnosis is however a fairly common event in literature. An anamnesis suggesting probable aspiration in a child under the age of 3 should direct doctors towards diagnostic and operative endoscope examinations of the patient, even where there is a negative clinical and radiological picture. Organic material, mainly peanuts, represented 60-75% of the findings, particularly in the 0-3 year age-band. In the other of cases inorganic material was extracted from school-age children. Aspiration of a FB exposes the patient to risk of serious complications and sequelae. Antibiotic, dexamethasone therapy and the ventilation support in the CPAP helped to avoid post-extractive sequelae. Prevention should in any case be the primary aim as regards to aspiration of foreign bodies in children. This should be stimulated by appropriate educational campaigns to raise awareness. The study included 62 child patients observed in the Department of Anesthesia and Intensive Care of the S.Orsola-Malpighi Hospital of Bologna over the last 11 years who were admitted for suspected FB aspiration.

[Infusion constituents in pediatric anesthesia]. / Apporti infusionali in anestesia pediatrica.

A suitable perioperative fluid therapy during paediatric anaesthesia presupposes a valuation of renal function and the preoperative fluid and electrolyte imbalance, a precise knowledge of fluid requirements and the physiological stress responses to surgery in different paediatric groups. Fluid administration must be suited to the pathology of the patients and surgical approach. It must replace the deficits from the preoperative status (including fasting), provide maintenance fluid and correct intraoperative translocated fluids and blood loss. Fluid management requires reduced hypotonic solution in order to prevent hyponatremia and avoid excessive amount of glucose which can be harmful. In order to prevent the dangers from blood transfusions you need to estimate the intraoperative loss and follow the "acceptable hematocrit" values. Rational intraoperative fluid management reduces perioperative morbidity and mortality.