Examining the viability of dorsal fin pigmentation for individual identification of poorly-marked delphinids.

Dolphin photo-identification has traditionally relied only on distinctive markings on the dorsal fin-this is problematic for delphinids whose populations exhibit a low mark ratio. We used common dolphins (genus Delphinus) as a model species to assess the viability of using pigmentation for photo-identification. Using a photo-identification catalogue of 169 adult individuals collected between 2002 and 2013, we extracted features that quantified pigmentation in a manner that was robust to lighting artefacts and dorsal fin orientation. We determined the proportion of individuals which exhibited pigmentation and examined temporal stability by (i) visually examining individuals and (ii) testing for seriation. We found 88-91% of images could be manually matched to the correct individual in the catalogue based on pigmentation patterns alone. A linear discriminant analysis classifier correctly identified the correct individual 77% of the time. We found 95% common dolphins exhibited distinctive pigmentation-all of which were temporarily stable. Our work challenges the current thinking that pigmentation is an unreliable feature for delphinid photo-identification and suggests that this feature could be applied to common dolphins and other poorly-marked delphinids.

Does using testicular sperm retrieval rather than ejaculated spermatozoa improve reproductive outcomes in couples with previous ART failure and poor ovarian response? A case-controlled study.

The objective of this study was to assess whether testicular-retrieved spermatozoa improve reproductive outcomes compared to fresh ejaculate in women with poor ovarian response and a history of previous ART failure. The study was performed as a retrospective case-control study at a university-based reproductive center in Montreal, Quebec, Canada. Eighteen poor-responder patients were matched 3 : 1 with 54 controls. Poor responders were defined as those with ≤3 oocytes retrieved at oocyte pickup. Cases were identified as poor responders, and only those with previous IVF failure(s) as an indication for testicular-retrieved spermatozoa were included. Controls were age and cycle attempt number matched. All patients were included only once. From January 1, 2009 to December 31, 2015, all patients and controls underwent an IVF cycle using ICSI with either testicular spermatozoa or ejaculated spermatozoa, respectively. Outcomes included live birth rate, pregnancy rate, miscarriage rate, oocyte number, and embryo transfer (ET) day. The results showed live birth rates, pregnancy rates, and miscarriage rates were similar. There were fewer day 2 ETs (8.5% vs. 48.6%, p = 0.01) and more day 5 blastocyst transfers (25.0% vs. 5.4%, p = 0.05) in the testicular sperm retrieval group compared to controls and thus an overall suggestion of better embryo quality in the testicular sperm group. Overall, however, the use of testicular sperm retrieval appears to add little. Women with poor ovarian response typically have a poor prognosis with respect to live birth rates, and this is further supported in this study. The suggestion of better embryo quality in the testicular-retrieved sperm group would need to be further assessed in a larger multicentered study.

The relationship between fat and progesterone, estradiol, and chorionic gonadotropin levels in Quebec cow's milk.

PURPOSE: The majority of milk in industrialized countries is obtained from pregnant cows, which contains increased levels of estrogen and progesterone compared to non-pregnant cows. The aim of this study was to quantify the amount of hormones present in milk with different fat content because previous studies on humans have shown potential effects of increased milk consumption on serum and urine hormone levels as well as on sperm parameters. However, it is unclear whether consumption of milk at the currently recommended levels would lead to systemic effects. METHODS: Samples of cow's milk of varying fat concentrations (0, 1, 2, 3.25, 10, and 35%) were analyzed via competitive ELISA assays. RESULTS: Progesterone concentrations were significantly correlated to increasing fat content of milk (r = 0.8251, p = 0.04). CONCLUSIONS: Research on conditions in which additional progesterone may have an effect on human health should consider inclusion of limitation of milk intake and its effects. Further studies are needed to determine the concentration of progesterone in milk of different fat content in other regions and countries and to quantify the potential pathophysiologic role.

Successful haploidentical donor hematopoietic stem cell transplant and restoration of STAT3 function in an adolescent with autosomal dominant hyper-IgE syndrome.

PURPOSE: Autosomal dominant hyper-IgE syndrome (AD-HIES), caused by mutations in Signal Transducer and Activator of Transcription 3 (STAT3) is associated with defective STAT3 signaling and Th17 differentiation and recurrent bacterial and fungal infections. Most patients suffer significant morbidity and premature mortality. Hematopoietic stem cell transplantation (HSCT) has been reported in a small number of cases, with mixed outcomes. We report successful haploidentical donor HSCT in a patient with AD-HIES. METHODS: Evaluation of lymphocyte subsets, STAT3 signaling, and Th17 cells was performed pre- and post-HSCT. RESULTS: A 14-year old female with AD-HIES developed recurrent methicillin-resistant Staphylococcus aureus (MRSA) abscesses. Immunologic analysis showed elevated IgE (4331 kU/L), absent Th17 cells, and markedly decreased STAT3 phosphorylation in cytokine stimulated peripheral blood mononuclear cells. She had breakthrough abscesses despite clindamycin and trimethoprim-sulfamethoxazole prophylaxis, and developed steroid refractory autoimmune hemolytic anemia. She underwent T-cell depleted haploidentical HSCT from her father following reduced intensity conditioning. She developed one MRSA hand abscess after transplant. Twenty-four months post transplant, she had complete donor chimerism (>95 % donor), normal absolute T cell numbers, and a normal percentage of Th17 cells. IgE was normal at 25 kU/L. She remains well 42 months after transplantation off all antibacterial prophylaxis. CONCLUSIONS: Haploidentical HSCT led to successful bone marrow engraftment, normalization of STAT3 signaling in hematopoietic cells, normalization of IgE, and restoration of immune function in this patient with AD-HIES.

Surface modification of PTMSP membranes by plasma treatment: Asymmetry of transport in organic solvent nanofiltration.

For the first time, the effect of asymmetry of the membrane transport was studied for organic solvents and solutes upon their nanofiltration through the plasma-modified membranes based on poly(1-trimethylsilyl-1-propyne) (PTMSP). Plasma treatment is shown to provide a marked hydrophilization of the hydrophobic PTMSP surface (the contact angle of water decreases from 88 down to 20°) and leads to the development of a negative charge of -5.2 nC/cm(2). The XPS measurements prove the formation of the oxygen-containing groups (Si-O and C-O) due to the surface modification. The AFM images show that the small-scale surface roughness of the plasma-treated PTMSP sample is reduced but the large-scale surface heterogeneities become more pronounced. The modified membranes retain their hydrophilic surface properties even after the nanofiltration tests and 30-day storage under ambient conditions. The results of the filtration tests show that when the membrane is oriented so that its modified layer contacts the feed solution, the membrane permeability for linear alcohols (methanol-propanol) and acetone decreases nearly two times. When the modified membrane surface faces the permeate, the membrane is seen to regain its transport characteristics: the flux becomes equal to that of the unmodified PTMSP. The well-pronounced effect of the transport asymmetry is observed for the solution of the neutral dye Solvent Blue 35 in methanol, ethanol, and acetone. For example, the initial membrane shows the negative retention for the Solvent Blue 35 dye (-16%) upon its filtration from the ethanol solution whereas, for the modified PTMSP membrane, the retention increases up to 17%. Various effects contributing to the asymmetry of the membrane transport characteristics are discussed.

Haploidentical related-donor hematopoietic cell transplantation in children using megadoses of CliniMACs-selected CD34(+) cells and a fixed CD3(+) dose.

We conducted a prospective phase II trial utilizing the CliniMACs system to perform CD34(+)-cell selection of PBSCs from haploidentical donors to evaluate engraftment and hematoimmunological reconstitution. In total, 21 children with hematological malignancies or nonmalignant conditions underwent conditioning with 1200 cGy TBI, thiotepa, fludarabine and Thymoglobulin. Patients received megadoses of CD34(+) cells (median: 22 × 10(6)/kg) with a fixed dose of 3 × 10(4)/kg CD3(+) cells/kg, and engraftment occurred in 90% with prompt recovery of neutrophils and platelets. Grade II acute GVHD (aGVHD) was seen in 32% (95% confidence interval (CI), 15-54%) of evaluable patients, there was no grade III-IV aGVHD, and chronic extensive GVHD was seen in 35% (95% CI, 17-59%) of patients. The estimated 2-year EFS was 62% (95% CI, 48-83%) with a median survivor follow-up of 49 months (range: 18-119 months). Patients with nonmalignant diseases had an estimated 2-year EFS of 100% (95% CI, 56-100%) and patients with malignancies in remission had an estimated 2-year EFS of 56% (95% CI, 22-89%). Megadose CD34(+) cells with a fixed CD3(+) cell dose from haploidentical related donors resulted in good outcomes for pediatric patients with nonmalignant diseases and those with malignant diseases transplanted in remission.

Anti-CD13 Abs in children with extensive chronic GVHD and their relation to soluble CD13 after allogeneic blood and marrow transplantation from a Children's Oncology Groups Study, ASCT0031.

Our group previously demonstrated a strong association between elevated plasma soluble CD13 enzyme activity and newly diagnosed extensive chronic GVHD (cGVHD) in children. As cytotoxic anti-CD13 Abs have been documented after blood and marrow transplant (BMT) in association with CMV infection and cGVHD, we hypothesized that soluble CD13 contributes to cGVHD pathogenesis by induction of CD13 reactive Abs and that anti-CD13 Abs could be additional biomarkers for newly diagnosed pediatric extensive cGVHD. Using prospectively collected plasma samples from pediatric allogeneic BMT (allo-BMT) subjects with cGVHD and controls without cGVHD enrolled in a large multi-institution Children's Oncology Group cGVHD therapeutic trial, we evaluated whether soluble CD13 correlates with induction of anti-CD13 Abs. We found that CD13 reactive Abs are present in a proportion of patients after allo-BMT, but did not seem to correlate with the presence of soluble CD13. Anti-CD13 Abs also did not meet our criteria as a diagnostic biomarker for cGVHD. These data do not confirm that induction of CD13 reactive Abs is a mechanism for cGVHD in children nor are part of the pathogenesis of cGVHD associated with elevated soluble CD13. The exact role of CD13 in cGVHD remains to be determined.

Clinical and immunologic outcomes following haplocompatible donor lymphocyte infusions.

We retrospectively analyzed the characteristics of 16 consecutive pediatric patients who received one or more G-CSF-mobilized donor lymphocyte infusions (DLI) following a T-cell-depleted haplocompatible hematopoietic SCT (HSCT) to enhance immune recovery and/or treat an infection. The median time from HSCT to administration of first DLI was 12 weeks and the median dose of DLI administered was 3 x 10(4)/kg (range, 2.5-6 x 10(4)/kg). The incidence of Grade I-II acute GVHD was 19% (95% confidence interval (CI), 6-44%), and there were no cases of Grade III-IV acute GVHD. Chronic GVHD developed in 13% (95% CI, 2-37%) of patients. In surviving patients who did not undergo a second stem cell infusion, T-cell numbers and function increased to a protective level in a median of 3 months (range, 2-12.5 months) following the first DLI administration. In patients given DLI for treatment of an infection, 75% (95% CI, 46-92%) cleared their infection after a median of 9 weeks (range, 1-27 weeks). In patients with CMV infection, the development of CMV-specific T cells was observed following DLI. The 1-year overall survival following haplocompatible DLI was 71% (95% CI, 59-83%), with a median follow-up of 16 months from the first DLI.

Infliximab for GVHD therapy in children.

GVHD remains a significant complication of allogeneic hematopoietic stem cell transplantation. Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of GVHD pathogenesis. Infliximab inhibits the binding of TNF-alpha with its cellular receptors and has been associated with encouraging responses in adults with severe GVHD. We retrospectively evaluated the efficacy and safety of infliximab 10 mg/kg i.v. once a week for a median of eight doses (range 1-162) in 24 children with steroid-resistant GVHD. The overall response rate in 22 evaluable children was 82% (12 CR+6 PR). Among those patients with acute GVHD, both skin and gastrointestinal involvement responded well to infliximab; however long-term outcome was poor. While infliximab may be useful to acutely control GVHD manifestations, GVHD recurs commonly upon discontinuation of infliximab. Within 100 days of the final infliximab dose, 77% of patients had bacterial infections, 32% had viral infections and 13.6% had probable or proven non-candidal invasive fungal infections. Infliximab appears to be well-tolerated and to have activity in steroid-resistant GVHD. Controlled studies to assess the pharmacokinetics and most effective dosing regimen of infliximab for the treatment of GVHD are warranted.

Circumpolar maternal blood contaminant survey, 1994-1997 organochlorine compounds.

During the past 20 years a number of studies have found neurological and immunological effects in the developing fetus and infants exposed to background or only slightly elevated levels of persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs). To address concerns arising from possible increased human exposure in the Arctic and possible effects of POPs, all circumpolar countries agreed in 1994 to monitoring of specific human tissues for contaminants in the Arctic under the Arctic Monitoring and Assessment Program (AMAP). Mothers in eight circumpolar countries contributed blood samples that were analysed at a single laboratory for 14 PCB congeners (IUPAC No. 28, 52, 99, 105, 118, 128, 138, 153, 156, 170, 180, 183, 187) and 13 organochlorine pesticides (aldrin, beta-hexachlorocyclohexane (beta-HCH), dichlordiphenyltrichloroethane (p,p'-DDT), diphenyldichloroethylene (p,p'-DDE), dieldrin, heptachlorepoxide, hexachlorobenzene (HCB), mirex, and the chlordane derivatives alpha-chlordane, gamma-chlordane, cis-nonachlor, oxychlordane and trans-nonachlor). Inuit mothers from Greenland and Canada have significantly higher levels of oxychlordane, transnonachlor and mirex than mothers from Norway, Sweden, Iceland and Russia. Inuit mothers from Greenland also have significantly higher levels of these contaminants than Inuit mothers from Canada and Alaska. These differences among Inuit groups may represent regional dietary preferences or different contaminant deposition patterns across the Arctic. Levels of PCBs are also elevated among some arctic populations due to their consumption of marine mammals and are in the range where subtle effects on learning and the immune system have been reported. The Russian mothers who consume mainly food imported from southern Russia have elevated levels of DDT, DDE, beta-HCH and a higher proportion of lower chlorinated PCB congeners. This study has allowed an assessment of the variation of contaminants such as PCBs and various organochlorine pesticides (DDT, chlordane, etc.) in human populations around the circumpolar north.

Effect of in vivo lymphocyte-depleting strategies on development of lymphoproliferative disorders in children post allogeneic bone marrow transplantation.

T cell depletion (TCD) of marrow is a proven method of graft-versus-host disease (GVHD) prophylaxis in allogeneic bone marrow transplantation (BMT). Nonetheless, TCD is associated with an increased risk of developing post transplant lymphoproliferative disorder (PTLD). Between 1986 and 1998, 241 pediatric patients at the University of Iowa underwent BMT using ex vivo TCD of marrow from mismatched related or matched unrelated donors. Additional GVHD prophylaxis included antithymocyte globulin (ATG) or anti lymphocyte globulin (ALG) post transplant (in vivo TCD). A total of 30 cases of PTLD were identified based upon a combination of clinical, histological, and immunological features. Nearly all cases occurred within 3 months post BMT. A statistically significant increase in PTLD incidence was noted for patients treated with ATG vs ALG (33 vs 9%). While grade I-II acute GVHD was more common in patients receiving ATG vs ALG, no difference in grade III-IV GVHD or overall survival was noted between the two groups. Assessment of immune recovery at various times post BMT revealed significantly fewer T cells in the ATG-treated group, suggesting the deleterious effect of ATG may be due to excessive depletion of donor-derived Epstein-Barr virus-specific cytotoxic T cells. Thus, caution should be exercised in the use of anti-T-cell antibody therapy for additional GVHD prophylaxis in the setting of TCD BMT.

Evaluation for synergistic suppression of T cell responses to minor histocompatibility antigens by chloroquine in combination with tacrolimus and a rapamycin derivative, SDZ-RAD.

The 4-aminoquinolines, chloroquine and hydroxychloroquine, can suppress chronic graft-versus-host disease (GVHD) following blood and marrow transplantation (BMT) in mice and humans, respectively. We hypothesized that chloroquine in combination with tacrolimus and the rapamycin derivative SDZ-RAD can synergistically suppress T cell responses and antigen-presenting cell (APC) function in vitro. We used the APC-dependent C57BL/6 anti-BALB.B T cell response and APC-independent anti-CD3epsilon antibody-induced response to evaluate the role of synergism between chloroquine and tacrolimus or SDZ-RAD on each component of a T cell response to minor histocompatibility antigens. We found that chloroquine with tacrolimus had a greater synergistic suppression of APC-dependent compared to the APC-independent T cell responses, with a combination index (CIx) for 50% inhibition by mean effect analysis of 0.16 and 0.50, respectively (a lower number indicates greater suppression). By contrast, chloroquine with SDZ-RAD had a similar CIx between the two responsed 0.50 vs0.45) suggesting only T cell suppression. Synergy between chloroquine and SDZ-RAD involved a direct effect on T cell cytokine production, whereas synergism between chloroquine and tacrolimus was due to an effect on both T cells and APCs. We conclude that the renal-sparing 4-aminoquinolines may be used syneristically with immunosuppressive drugs currently used for BMT.

Iodine fortification is related to increased weight-for-age and birthweight in children in Asia.

Severe iodine deficiency causes stunting and mental retardation in utero, but the relation between mild deficiency and child growth is not well known. The use of iodated salt in relation to anthropometric data was examined from recent survey data. After potential confounding factors had been controlled for, significant associations were seen in Bangladesh, India, Nepal, and Sri Lanka. The use of iodated salt was related to increased weight-for-age and mid-upper-arm circumference, most strongly in the second year of life, mainly affecting soft tissue (thinness). The relation with weight-for-age was greater among children of mothers with lower body mass index. The use of iodated salt was related to birthweight in Sri Lanka and in the Philippines, where iodized oil capsules given during pregnancy had a negative effect when used with high levels of iodine in salt. The associations generally were concentrated in large geographic areas, possibly because of interactions with other environmental factors (e.g., selenium and arsenic). The apparent growth response to iodine may reflect functional effects of mild deficiency, which is widespread, possibly including effects on brain development.

Cross talk: interview with Al Gilman.

Alfred Goodman Gilman was born in the same year (1941) that his father and Louis Goodman published the first edition of The Pharmacological Basis of Therapeutics. Pharmacology has thus always been part of his life, and in his own career he has focused primarily on cell signaling. For the past twenty years, he has chaired the Department of Pharmacology at UT Southwestern, and his long list of accomplishments includes a Nobel Prize (1994) for his work on G proteins. In 1998, Gilman embarked on his most ambitious program of research yet, bringing dozens of leading investigators from the cell signaling community to Dallas in order to plan out a ten-year project aiming "to understand as completely as possible the relationships between sets of inputs and outputs in signaling cells." Now directing the full-fledged, federally funded Alliance for Cellular Signaling, Gilman stresses that a solid database for constructing a "virtual cell" will depend on extensive collaboration from the entire signaling community. (For a complete Program Summary, and to register for membership in the Alliance, consult www.cellularsignaling.org.) The luminaries that were invited to the Dallas planning meeting, in fact, were greeted at the door with a note from Gilman exhorting them: please check EGO at door.

Molecular basis for P-site inhibition of adenylyl cyclase.

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.

Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase.

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G(s)alpha. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C(1) and C(2)) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the beta4-beta5 loop of the C(2) domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C(1) domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C(2) domain of adenylyl cyclase for the wild type C(1) domain was exploited to isolate a complex containing VC(1), IIC(2), and G(s)alpha-guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in the absence of forskolin and a complex of VC(1), IIC(2), forskolin, and P-site inhibitor in the absence of G(s)alpha-GTPgammaS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G(s)alpha.