Pancreas-After-Islet Transplantation in Nonuremic Type 1 Diabetes: A Strategy for Restoring Durable Insulin Independence.

Islet transplantation offers a minimally invasive approach for ß cell replacement in diabetic patients with hypoglycemic unawareness. Attempts at insulin independence may require multiple islet reinfusions from distinct donors, increasing the risk of allogeneic sensitization. Currently, solid organ pancreas transplant is the only remaining surgical option following failed islet transplantation in the United States; however, the immunologic impact of repeated exposure to donor antigens on subsequent pancreas transplantation is unclear. We describe a case series of seven patients undergoing solid organ pancreas transplant following islet graft failure with long-term follow-up of pancreatic graft survival and renal function. Despite highly variable panel reactive antibody levels prior to pancreas transplant (mean 27 ± 35%), all seven patients achieved stable and durable insulin independence with a mean follow-up of 6.7 years. Mean hemoglobin A1c values improved significantly from postislet, prepancreas levels (mean 8.1 ± 1.5%) to postpancreas levels (mean 5.3 ± 0.1%; p = 0.0022). Three patients experienced acute rejection episodes that were successfully managed with thymoglobulin and methylprednisolone, and none of these preuremic type 1 diabetic recipients developed stage 4 or 5 chronic kidney disease postoperatively. These results support pancreas-after-islet transplantation with aggressive immunosuppression and protocol biopsies as a viable strategy to restore insulin independence after islet graft failure.

Metallothionein as a clonable tag for protein localization by electron microscopy of cells.

A benign, clonable tag for the localization of proteins by electron microscopy of cells would be valuable, especially if it provided labelling with high signal-to-noise ratio and good spatial resolution. Here we explore the use of metallothionein as such a localization marker. We have achieved good success with desmin labelled in vitro and with a component of the yeast spindle pole body labelled in cells. Heavy metals added after fixation and embedding or during the process of freeze-substitution fixation provide readily visible signals with no concern that the heavy atoms are affecting the behaviour of the protein in its physiological environment. However, our methods did not work with protein components of the nuclear pore complex, suggesting that this approach is not yet universally applicable. We provide a full description of our optimal labelling conditions and other conditions tried, hoping that our work will allow others to label their own proteins of interest and/or improve on the methods we have defined.

Vascular neoplasm or pseudovascular nevus? Potential pitfalls in diagnosis.

Melanocytic nevi, on histopathologic evaluation, occasionally contain slit-like clefts or spaces that may resemble vascular or lymphatic spaces. The spaces may contain blood or, perhaps more concerning, nests of melanocytes that could suggest lymphatic invasion of melanoma. When lined by melanocytes rather than true endothelium, these pseudovascular spaces within melanocytic nevi are generally attributable to tissue processing artifact. When the space in question is pronounced, a proper diagnostic work-up is prudent in order to exclude a true vascular neoplasm or melanoma. In this case series we present several melanocytic lesions with prominent vascular-appearing spaces that warranted further investigation.

African origin of an intragenic deletion of the human P gene in tyrosinase positive oculocutaneous albinism.

Oculocutaneous albinism (OCA) is a genetically heterogeneous hypopigmentation disorder. One of the two major autosomal recessive forms involves the tyrosinase gene (OCA1), while the other form (OCA2) has recently been associated with alterations of the P gene on chromosome 15. OCA2 is about twice as common as OCA1 in African and African-American populations. We now describe an interstitial deletion that removes a single exon of the P gene. In a large family from an inbred population of tri-racial origin, all individuals with OCA2 were found to be homozygous for this allele. Moreover, the same mutant P allele was detected in several unrelated African American individuals with OCA2, but not in Caucasians with OCA2. The detection of the same allele in two unrelated Africans with OCA2 indicates an African origin for this allele.

Effects of motion and audio-visual redundancy on upright and inverted face and feature preferences in 4-13-month old pre- and full-term NICU graduates.

NICU infants are reported to have diminished social orientation and increased risk of socio-communicative disorders. In this eye tracking study, we used a preference for upright compared to inverted faces as a gauge of social interest in high medical risk full- and pre-term NICU infants. We examined the effects of facial motion and audio-visual redundancy on face and eye/mouth preferences across the first year. Upright and inverted baby faces were simultaneously presented in a paired-preference paradigm with motion and synchronized vocalization varied. NICU risk factors including birth weight, sex, and degree of CNS injury were examined. Overall, infants preferred the more socially salient upright faces, making this the first report, to our knowledge, of an upright compared to inverted face preference among high medical risk NICU infants. Infants with abnormalities on cranial ultrasound displayed lower social interest, i.e. less of a preferential interest in upright faces, when viewing static faces. However, motion selectively increased their upright face looking time to a level equal that of infants in other CNS injury groups. We also observed an age-related sex effect suggesting higher risk in NICU males. Females increased their attention to the mouth in upright faces across the first year, especially between 7-10 months, but males did not. Although vocalization increased diffuse attention toward the screen, contrary to our predictions, there was no evidence that the audio-visual redundancy embodied in a vocalizing face focused additional attention on upright faces or mouths. This unexpected result may suggest a vulnerability in response to talking faces among NICU infants that could potentially affect later verbal and socio-communicative development.

Fibronectin expression during myogenesis.

The biosynthesis and localization of fibronectin during chick muscle differentiation are described. This study employed two monoclonal antibodies, one that selectively killed mononucleated cells and one specific for avian fibronectin. These antibodies allowed precise analyses of fibronectin expression in well-defined cultures of myoblasts or myotubes and avoided the complications of exogenous fibronectin and contamination by fibroblasts or unfused myoblasts. Fibronectin synthesis, as a fraction of total protein synthesis, remains constant at 0.3-0.4% before and after myoblast fusion, suggesting that the absolute rate of fibronectin synthesis may increase somewhat when myotubes synthesize and accumulate myofibrillar proteins. The pattern of fibronectin arrangement does change during myogenesis. In myotube cultures, the appearance of pulse-labeled fibronectin at the cell surface and its secretion into the medium begin after a 2-3-h lag period, in contrast to the 30-min lag period observed in fibroblast cultures. This lag between polypeptide biosynthesis and the exteriorization of the new protein is thus a characteristic of each cell type rather than the protein. All of the major secretory proteins of myogenic cells, including fibronectin and collagenous components, share this 2-3-h intracellular transit time.

The mouse pink-eyed dilution gene: association with human Prader-Willi and Angelman syndromes.

Complementary DNA clones from the pink-eyed dilution (p) locus of mouse chromosome 7 were isolated from murine melanoma and melanocyte libraries. The transcript from this gene is missing or altered in six independent mutant alleles of the p locus, suggesting that disruption of this gene results in the hypopigmentation phenotype that defines mutant p alleles. Characterization of the human homolog revealed that it is localized to human chromosome 15 at q11.2-q12, a region associated with Prader-Willi and Angelman syndromes, suggesting that altered expression of this gene may be responsible for the hypopigmentation phenotype exhibited by certain individuals with these disorders.

Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.

Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined the oral and gut microbiome of melanoma patients undergoing anti-programmed cell death 1 protein (PD-1) immunotherapy (n = 112). Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus nonresponders. Analysis of patient fecal microbiome samples (n = 43, 30 responders, 13 nonresponders) showed significantly higher alpha diversity (P < 0.01) and relative abundance of bacteria of the Ruminococcaceae family (P < 0.01) in responding patients. Metagenomic studies revealed functional differences in gut bacteria in responders, including enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplants from responding patients. Together, these data have important implications for the treatment of melanoma patients with immune checkpoint inhibitors.

Determinants of aggressive and prosocial behaviour among Jamaican schoolboys.

PURPOSE: This study examines risk factors for aggression among boys in Kingston, Jamaica. METHODS: One hundred and one aggressive and 101 prosocial schoolboys in grades 5-6 (mean age 11.7, SD 0.6 years) were selected by peer and teacher ratings from 10 schools in the capital city, Kingston, during 1998. They were given in-depth questionnaires, arithmetic, reading and verbal intelligence tests and their behaviour was rated. Their parents were also given a detailed questionnaire. RESULTS: The aggressive boys reported significantly more involvement in fights than the prosocial boys. They had lower scores on spelling/reading and verbal IQ, less ambitious aspirations and poorer quality school uniforms. They were not more likely to infer hostile intent in ambiguous situations but were more likely to respond with aggression. Aggressive boys came from poorer homes with more marijuana use, less parental affection or supervision and more family discord. They were less exposed to religious instruction, their parents had lower occupational levels and were more likely to be in common-law unions than married. They were more exposed to neighbourhood violence and were punished more often at home and at school. Logistic regression analyses were carried out to determine the independent risk factors for aggression. Exposure to neighbourhood violence, physical punishment at home and family discord were associated with increased risk; parents' being married, practising religion as a family and better school uniforms were associated with reduced risk. CONCLUSIONS: Although community violence was a serious problem, family characteristics were also important risk factors for aggressive behaviour.

Fine social aspiration: Twitter as a voice for cytopathology.

Social media is an influential tool that has the power to transform cytopathology. Twitter is being used more and more to share cutting-edge updates from pathology meetings ("live-tweeting"). Modern smartphones can now take high resolution microscopic photographs and easily transmit them worldwide via Twitter, Facebook, and other social media, allowing cytopathologists to share educational pearls and discuss difficult cases on a global scale like never before. Social media also allows cytopathologists to share a behind-the-scenes look at their subspecialty with other physicians and even the non-medical public, helping them to better understand the crucial importance of cytopathology in modern medicine. This could positively impact rapport with other specialties, influence policy making, and possibly even improve delivery of patient care. Rare disease patient communities are being formed by patients on Facebook. By joining and volunteering with these patient groups, cytopathologists would have further opportunity to interact directly with patients and their family members, explaining the role of cytopathology in patient care and helping patients to better understand their own diseases. Social media enables cytopathologists and their colleagues in other pathology subspecialties to easily and rapidly form a broad and diverse worldwide network with one another. The authors believe that this is the key to a bright future for our specialty, a strong unified global community of pathologists all working together for education, patient advocacy, and outstanding patient care. Social media can allow us to build that community, strengthen its bonds, and harness its power like never before in history. Diagn. Cytopathol. 2017;45:705-713. © 2017 Wiley Periodicals, Inc.

Effect of accommodating sucking and nosing on the behaviour of artificially reared piglets.

Neonatal piglets are often used in biomedical research applications that require artificial rearing. Social housing can be problematic because the piglets develop belly nosing, navel and ear sucking that can result in injury. Our objective was to determine the effectiveness of using feeding devices that provide various opportunities for sucking and nosing behaviour on reducing piglet-directed behaviour of group-housed laboratory piglets. Fifteen piglets were used in each of four trials. The piglets nursed their dam for approximately 72 h to obtain passive immunity before transfer to a laboratory facility where they were allotted, five per group, to one of three stainless steel isolator units. Each unit featured a different style of feeding system for the delivery of milk replacer: a plastic trough (T), a nipple (N) mounted on a smooth plexiglass wall, or a nipple mounted on a pliant bag of sterile water (artificial udder [AU]). Each system had five feeding spaces so that all piglets fed simultaneously. Milk was provided at 6-h intervals, and behaviour was recorded on alternate days for 12 days post-weaning. Although trough-fed piglets began to eat much sooner than those piglets fed from nipples, time spent nosing, chewing or sucking on pen-mates and belly nosing were markedly higher in T piglets than in either N or AU, overall (mean: P<0.05) and over time (quadratic: P<0.05). Over time, N piglets developed a stereotypic snout rubbing on the wall behind the nipples, while AU piglets massaged and often fell asleep in contact with the udder from day 2 of the trial. Resting patterns were also affected. N and AU piglets settled down to rest more quickly (P<0.01) and spent significantly more time resting in the hour following feeding than T piglets (P<0.05). A feeding device that accommodates both sucking and massage can significantly reduce piglet-directed behaviour and may facilitate social housing of artificially reared piglets.

High-level tissue-specific expression of functional human factor VIII in mice.

Hemophilia A results from subnormal levels of blood coagulation factor VIII (FVIII) and is an attractive target for gene therapy. However, progress has been impeded by features of FVIII biology such as low mRNA accumulation and the instability of the protein. We have shown previously that a FVIII adenoviral vector, Av1ALH81, allowed high-level expression of human FVIII in mice sustained for several weeks. Here, we have generated a second FVIII adenoviral vector, Av1ALAPH81, in which an intron was introduced into the FVIII expression cassette. Administration of Av1ALAPH81 to mice resulted in significantly increased FVIII plasma levels, 1,046 +/- 163 ng/ml compared to 307 +/- 93 ng/ml of FVIII detected in mice that received Av1ALH81. Normal FVIII levels in humans are 100-200 ng/ml and therapeutic levels are as low as 10 ng/ml. Therapeutic levels are defined as the amount of FVIII necessary to convert severe hemophilia to a moderate or mild hemophiliac condition. The increased potency of the second FVIII adenoviral vector allowed the administration of significantly lower, less toxic vector doses, while retaining the potential for high FVIII expression. Furthermore, we demonstrate that adenoviral-mediated expression of human FVIII can be limited to the liver by inclusion of a liver-specific promoter, thereby achieving the first step in regulated expression of human FVIII in vivo.

In vivo gene delivery and expression of physiological levels of functional human factor VIII in mice.

Hemophilia A is caused by blood coagulation factor VIII (FVIII) deficiency and is an attractive target for gene therapy. However, features of FVIII physiology, such as the instability of the mRNA and protein, have provided obstacles to the design of a feasible strategy for the transfer and expression of the human FVIII gene in vivo. We have constructed a recombinant adenoviral vector, Av1ALH81, that contains the human FVIII cDNA from which the B-domain has been deleted (BDD FVIII) and extensively characterized this vector in vitro and in vivo. In vitro, HepG2, human hepatoma cells, transduced with Av1ALH81 secreted high levels of biologically active human BDD FVIII measured by the Coatest bioassay (> 2,400 mU per 10(6) cells per 24 hr). Administration of Av1ALH81 to mice, via tail vein, resulted in expression of human BDD FVIII in the mouse plasma at levels averaging 307 +/- 93 ng/ml 1 week post-injection, measured by a sensitive human FVIII-specific ELISA. Normal FVIII levels in humans are 100-200 ng/ml, and therapeutic levels are as low as 10 ng/ml. Purification of the human FVIII from the mouse plasma, and subsequent Coatest analysis, revealed that the human FVIII produced in the mice was biologically active. In addition, the duration of FVIII expression in vivo was followed, and high-level FVIII expression was sustained over a period of several weeks. The finding that an adenoviral vector can mediate high-level expression of human FVIII in an animal model provides the basis for the development of gene therapy for hemophilia A.

Aberrant pH of melanosomes in pink-eyed dilution (p) mutant melanocytes.

In past studies, we cloned the mouse p gene and its human homolog P, which is associated with oculocutaneous albinism type 2. Both mouse and human genes are expressed in melanocytes and encode proteins predicted to have 12 membrane-spanning domains with structural homology to known ion transporters. We have also demonstrated that the p protein is localized to the melanosomal membrane and does not function as a tyrosine transporter. In this study, immunohistochemistry and confocal microscopy were used to show that the p protein plays an important role in the generation or maintenance of melanosomal pH. Melanosomes (and their precursor compartments) were defined by antiserum directed against the melanosomal marker tyrosinase related protein 1. Acidic vesicles were identified by 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine incorporation, visualized with anti-dinitrophenol. In C57BL/6+/+ (wild-type) melanocytes, 94.2% of vesicles demonstrated colocalization of tyrosinase related protein 1 and 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine, indicating that almost all melanosomes or their precursors were acidic. By contrast, only 7%-8% of the staining vesicles in p mutant cell lines (pJ/pJ and pcp/p6H) showed colocalization of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine. Thus, without a functional p protein, most melanosomes and their precursors are not acidic. As mammalian tyrosinase activity in situ is apparently dependent on low pH, we postulate that in the absence of a low pH environment brought about by ionic transport mediated by the p protein, tyrosinase activity is severely impaired, leading to the minimal production of melanin that is characteristic of p mutants. Additionally (or alternatively), an abnormal pH may also impair the assembly of the normal melanogenic complex.

Identification of a novel transcript produced by the gene responsible for the Hermansky-Pudlak syndrome in Puerto Rico.

Hermansky-Pudlak Syndrome (HPS) is a rare, autosomal recessive disorder that is characterized by oculocutaneous albinism, a predisposition to mild bleeding caused by storage-pool deficient platelets, and a ceroid storage disorder. A gene responsible for HPS in Puerto Rico maps to chromosome 10q2 and isolation of the gene has been reported. We have now identified a variant HPS cDNA that contains the same 5' sequence as the published HPS gene and a unique 3' sequence. Analysis of genomic DNA suggests that the two cDNA are derived from alternative transcripts of a single gene; two polyadenylated transcripts were found in normal human melanocytes, human bone marrow cells, human melanoma cells, lymphoblastoid cell lines, and megakaryocytic leukemia cells by reverse transcriptase polymerase chain reaction and northern analysis. The splicing exhibited by this gene is identical to the splicing found to produce two alternative transcripts of the Chediak-Higashi Syndrome gene, another pigment disorder exhibiting platelet storage pool deficiency. These studies show that the HPS gene on chromosome 10 is complex and may have more than one biologically active transcript.

Weaning-food viscosity and energy density: their effects on ad libitum consumption and energy intakes in Jamaican children.

The effects of weaning-food viscosity and energy density on consumption and energy intake were determined in 15 non-breast-fed Jamaican children aged 7-15 mo under standardized conditions. We tested whether feeding thick, energy-dense porridge four times daily resulted in increased energy intakes and whether amylase treatment to reduce viscosity offered any advantage. When a traditional liquid, low-density porridge (2.15 kJ/g) was fed, the mean (+/- SD) daily consumption was 139 +/- 25 g/kg and the mean daily energy intake was 296 +/- 54 kJ/kg. When a semisolid high-density porridge (4.09 kJ/g) was fed, consumption was significantly lower (98 +/- 21 g/kg) but the daily energy intake was significantly higher--402 +/- 85 kJ/kg (P < 0.001). Amylase treatment of the thick energy-dense porridge did not increase intakes further. Meal duration for the thick porridge (12.9 +/- 4.0 min) was significantly longer than that for the low-density (7.4 +/- 2.6 min) or amylase-treated (6.4 +/- 1.8 min) porridges.

Therapeutic effects of oral zinc in acute and persistent diarrhea in children in developing countries: pooled analysis of randomized controlled trials.

BACKGROUND: Zinc deficiency is prevalent in children in developing countries. Supplemental zinc provides therapeutic benefits in diarrhea. OBJECTIVE: We sought to measure the effect of supplemental zinc given with oral rehydration therapy during recovery from acute or persistent diarrhea. DESIGN: We conducted pooled analyses including all available published and unpublished randomized controlled trials of the effects of supplementary oral zinc in children aged <5 y with acute or persistent diarrhea. We used Cox survival regression analysis to evaluate the overall effect of zinc on continuation of diarrhea and possible differential effects in subgroups divided by sex, age, weight-for-height, and initial plasma zinc concentration. Dichotomous outcomes were analyzed by logistic regression. To assess the effects of excluding studies without original data from the pooled analyses, effect-size was estimated for all studies by using random-effects models. RESULTS: Zinc-supplemented children had a 15% lower probability of continuing diarrhea on a given day (95% CI: 5%, 24%) in the acute-diarrhea trials and a 24% lower probability of continuing diarrhea (95% CI: 9%, 37%) and a 42% lower rate of treatment failure or death (95% CI: 10%, 63%) in the persistent-diarrhea trials. In none of the subgroup analyses were the 2 subgroups of each pair significantly different from each other; however, in persistent diarrhea there tended to be a greater effect in subjects aged <12 mo, who were male, or who had wasting or lower baseline plasma zinc concentrations. CONCLUSION: Zinc supplementation reduces the duration and severity of acute and persistent diarrhea.

Analysis of bleeding patterns and resumption of fertility following discontinuation of a long acting injectable contraceptive.

PIP: To assess the effects of Depo-medroxyprogesterone acetate (DMPA) upon the menstrual patterns and resumption of fertility in women who have discontinued usage 53 patients who had previously received DMPA were studied. 26 of the 53 women had not used oral contraceptive steroids after discontinuing DMPA, the remaining 27 had used an oral contraceptive for varying amounts of time. 40 of the 53 women did not use an oral contraceptive prior to their initial bleeding episode after DMPA use. 48% of these women resumed normal cyclic bleeding. The remaining 21 all resumed bleeding within 6 months after DMPA discontinuation, but bleeding was irregular. 8 of the 21 had heavy flow, which was controlled with oral steroids. Of the total 53 patients, 36 did not begin the use of oral contraceptives for at least 6 months after cessation of DMPA usage. 50% of them had cycles. It was estimated that ovulation returned to 93% of the group of women who could be studied after discontinuing DMPA. It would seem that the duration of time to the onset of regular menses is not related to the number of injections received. Of the patients who conceived, the incicence of abnormal births did not differ from that of the general population. This drug seems to allow for normal endometrial response once the drug's effect has diminished. Since it is impossible to predict the time required for the drug's effect to diminish, it is recommended that the drug be given to women who are not spacing their family.^ieng

Sterol content of foods of plant origin.

Available data on phytosterols from the world's literature have been compiled and summarized. There still exists a paucity of data on the quantities of plant sterols in many foods. More extensive data are available on the relative sterol composition. Our compilation shows that plant oils are excellent sources of phytosterols. Nuts and seeds contain moderate levels, and fruits and vegetables generally contain the lowest concentrations of plant sterols. Analyses of the minor sterols, namely, the delta5- and delta7-phytosterols, have become available only recently.

Dietary intake and observed activity of stunted and non-stunted children in Kingston, Jamaica. Part II: Observed activity.

It is generally believed that activity levels are reduced in poorly nourished children. This may conserve energy, but may have a detrimental effect on mental development. However there are few data which support this hypothesis. No previous studies were found which looked at the activity levels of stunted children. In this study we modified the time and motion observation methods of Torun (1984) by using recording periods of 1 instead of 10 min. This significantly reduced the estimated time spent by young children in moderate and vigorous activities. Four hours of observing young children while they were awake, using the modified method, produced highly reliable data. The activity levels of a subsample of children described in part I were observed. The children were aged 12-24 months and were observed for 4 h on 2 d. Seventy-eight stunted children (height less than -2 SD of median, NCHS) were compared with 26 non-stunted children (height greater than -1 SD). The stunted children were significantly less active than the non-stunted children, although the difference in activity rating was small (3.4 per cent). They spent more time in light activities (P less than 0.001) and less time in moderate or vigorous activities (P less than 0.01). Both groups slept a similar amount. Using Torun's values for the energy cost of activities it appears unlikely that the reduced activity would conserve energy to a great extent, but it may still be of biological importance, since it is comparable to the energy cost of growth at this age. Activity levels increased with age but were not significantly related to weight for height.(ABSTRACT TRUNCATED AT 250 WORDS)