Successful reversal of streptozotocin-induced diabetes with stable allogeneic islet function in a preclinical model of type 1 diabetes.

The recent focus on islet transplantation as primary therapy for type 1 diabetes has heightened interest in the reversal of type 1 diabetes in preclinical models using minimal immunosuppression. Here, we demonstrated in a preclinical rhesus model a consistent reversal of all measured glycemic patterns of streptozotocin-induced type 1 diabetes. The model used single-donor islet transplantation with induction of operational tolerance. The term "operational tolerance" is used to indicate durable survival of single-donor major histocompatibility complex (MHC)-mismatched islet allografts without maintenance immunosuppressive therapy and without rejection or loss of functional islet mass or insulin secretory reserve. In this operational tolerance model, all immunosuppression was discontinued after day 14 posttransplant, and recipients recovered with excellent health. The operational tolerance induction protocol combined peritransplant anti-CD3 immunotoxin to deplete T-cells and 15-deoxyspergualin to arrest proinflammatory cytokine production and maturation of dendritic cells. T-cell deficiency was specific but temporary, in that T-cell-dependent responses in long-term survivors recovered to normal, and there was no evidence of increased susceptibility to infection. Anti-donor mixed lymphocyte reaction responses were positive in the long-term survivors, but all showed clear evidence of systemic T-helper 2 deviation, suggesting that an immunoregulatory rather than a deletional process underlies this operational tolerance model. This study provides the first evidence that operational tolerance can protect MHC nonhuman primate islets from rejection as well as loss of functional islet mass. Such an approach has potential to optimize individual recipient recovery from diabetes as well as permitting more widespread islet transplantation with the limited supply of donor islets.

A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities.

Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3epsilon scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH(2) terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3epsilongamma was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH(2) terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3epsilongamma. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3epsilongamma increased approximately 3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand-scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements.

Adaptive emergency scenery video communications using HEVC for responsive decision support in disaster incidents.

This study proposes a unifying framework for m-Health video communication systems that provides for the joint optimization of video quality, bitrate demands, and encoding time. The framework is video modality and infrastructure independent and facilitates adaptation to the best available encoding mode that satisfies underlying technology and application imposed constraints. The scalability of the proposed algorithm is demonstrated using different HEVC encoding configurations and realistic modelling of 802.11× wireless infrastructure for emergency scenery and response videos. Extensive experimentation shows that a jointly optimal solution in the encoding time, bitrate, and video quality space is feasible.

A novel mutation of the human luteinizing hormone receptor in 46XY and 46XX sisters.

We report a novel homozygous mutation of the LH receptor (LHR) gene in three siblings: two 46XY and one 46XX. The 46XY siblings presented with female external genitalia, primary amenorrhea, and lack of breast development. Hormonal evaluation revealed a markedly elevated LH level with a low testosterone level, which failed to increase after human CG stimulation. Enzymatic deficiencies of testosterone biosynthesis were eliminated as possible etiologies. Histologic analysis of the inguinal gonads in a 46XY sibling revealed no Leydig cells; Sertoli cells, spermatogonia, and primary spermatocytes were seen. The 46XX sibling had female external genitalia, normal breast development, and primary amenorrhea. Hormonal analyses showed markedly elevated LH levels and low plasma 17 beta-estradiol levels. Genetic analysis of the LHR revealed a homozygous missense mutation at exon 11 of the LHR gene. Guanine was replaced by adenine (GAA-->AAA), resulting in a substitution of lysine for glutamic acid (glu) at amino acid position 354 of the receptor. This mutation is located in the extracellular domain adjacent to the first transmembrane helix of the LHR. Glutamic acid at position 354 of the LHR has been highly conserved throughout evolution. Functional analysis of the LHR mutation, using an in vitro mutagenesis-transfection assay, demonstrated complete loss of function, indicated by the lack of cAMP production after human CG stimulation in transfected human embryonic kidney 293 cells. Screening of family members demonstrated heterozygosity for the mutation, indicating autosomal recessive inheritance. Delineation of the specific genetic defect in this family confirms recent reports that a single mutation in the LHR gene causes male pseudohermaphroditism in 46XY subjects and primary amenorrhea in 46XX subjects. More importantly, it also defines a new region of the LHR molecule that is critical for biologic activity.

Peritransplant tolerance induction in macaques: early events reflecting the unique synergy between immunotoxin and deoxyspergualin.

BACKGROUND: Day of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model. METHODS: Immunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined. RESULTS: Allografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG x 15 days, in 50% with DSG x 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation. CONCLUSIONS: The study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.

Diagnosis and management of growth hormone deficiency in adults.

In adults, GHD is a clinical syndrome that occurs in patients with pituitary or hypothalamic disease. It may be asymptomatic or present with relatively nonspecific constitutional symptoms. Most patients have abnormal body composition, consisting of increased fat mass and decreased lean mass. Life expectancy is significantly decreased in hypopituitary patients with GHD, with cardiovascular disease a common cause of death. Treatment with growth hormone reverses abnormalities in body composition and may reduce cardiovascular risk factors; however, the long-term treatment outcomes regarding mortality, the incidence of cardiovascular disease, bone fractures, tumor development, and recurrence are not known. Longer prospective clinical studies are needed. The major manufacturers of growth hormone have initiated postmarketing surveillance databases to monitor the safety of growth hormone treatment.

Jod-Basedow syndrome following oral iodine and radioiodinated-antibody administration.

This is a case of thyrotoxicosis, presumably due to Jod-Basedow syndrome, after stable iodine ingestion for thyroid blockade in a patient with ovarian carcinoma having 131I-labeled monoclonal antibody imaging. With the increased use of radioiodinated antibodies, for therapy and imaging, this possible side effect of excess stable iodine administration should be noted, especially in patients with pre-existing goiter.

Primate renal transplants using immunotoxin.

BACKGROUND: T-lymphocyte depletion 7 days before transplantation with immunotoxin FN 18-CRM9 has resulted in tolerance to subsequent renal allografts. We tested the effect of giving immunotoxin on the day of the transplantation and evaluated its effect on rhesus monkey and allograft survival, on antibody production, and on T-cell recovery. METHODS: Major histocompatibility complex mismatched renal allografts were performed in rhesus monkeys. Immunotoxin was given starting on the day of transplantation, with and without prednisone and mycophenolate mofetil for 3 days. T-cell subsets and alloantibody levels were measured by flow cytometry. The ability of treated monkeys to develop antibody to tetanus, diphtheria, and xenoantibody was measured. Histology of renal transplants was read in a blinded manner. RESULTS: Immunotoxin started on the day of transplantation resulted in prolonged allograft survival in all treatment groups. Graft loss between days 50 and 135 was most often due to interstitial nephritis. Later graft loss was due to chronic rejection. Monkeys had intact antibody responses to alloantigen, tetanus, diphtheria, and xenoantibody. Their CD4 cells recovered gradually over 6 months. CONCLUSIONS: Immunotoxin reliably prolongs renal allograft survival when started on the day of transplantation, but interstitial nephritis and chronic rejection limit the development of long-term tolerance. T-cell-dependent B-cell responses remain intact after treatment.

Rheumatic manifestations of pituitary tumors.

Pituitary tumors may cause rheumatologic problems as a result of under production or overproduction of one pituitary hormone. Excessive growth hormone causes destruction of cartilage by a direct action. Facial and acral changes and arthralgias may be some of the first symptoms of acromegaly. The arthritis associated with acromegaly is often devastating. Carpal tunnel syndrome is very common in patients with acromegaly. Adrenocorticotropin (ACTH) has indirect effects via the action of glucocorticoid on bones, muscles, and the immune system. Proximal muscle weakness is a characteristic feature of Cushing's syndrome. Patients with Cushing's syndrome commonly have osteopenia and osteoporosis that lead to an increase in bone fractures. Avascular necrosis is associated with exogenous steroid administration. The effects of too much glucocorticoid or too rapid withdrawal can be severe. Gonadotropins act via the gonadal steroids and protect bone mass from loss. Prolactin is less involved in rheumatologic disease; the data for which are limited in humans. Pituitary tumors can have manifestations similar to rheumatologic disorders and should be included in the differential diagnosis of these diseases.

Genetic construction and characterization of an anti-monkey CD3 single-chain immunotoxin with a truncated diphtheria toxin.

We have previously developed a chemically conjugated anti-rhesus monkey CD3 immunotoxin FN18-CRM9 that can deplete in vivo T cells and induce long term tolerance of mismatched renal allograft in rhesus monkeys. This immunotoxin is a monkey analogue of anti-human CD3 immunotoxin UCHT1-CRM9. In this study, we cloned the light and heavy chain variable regions of anti-monkey CD3 monoclonal antibody FN18 and constructed a single-chain Fv (sFv) by linking variable light and variable heavy regions with a (Gly4Ser)3 linker. The single-chain immunotoxin DT390-FN18sFv was constructed by ligating the sFv to the carboxyl terminus of DT390, a truncated form of diphtheria toxin. The DT390-FN18sFv fusion protein was expressed in Escherichia coli and purified with Ni-RTA affinity and anion exchange columns. Similar to the chemically conjugated immunotoxin FN18-CRM9, DT390-FN18sFv can also specifically inhibit protein synthesis in primary monkey T cells in a dose-dependent manner. DT390-FN18sFv at 10(-7) mol/L or FN18-CRM9 at 10(-8) mol/L is sufficient to reduce protein synthesis of monkey primary T cells to less than 5% of the control. The 50% inhibition dosage (IC50) of FN18-CRM9 is 1 x 10(-10) mol/L, while the IC50 of DT390-FN18sFv is 1 x 10(-8) mol/ L, reflecting the lowered affinity of monovalent Fab' FN18 to its parental divalent antibody. The availability of functional FN18sFv will provide the basis for the construction of divalent anti-CD3 immunotoxins for preclinical studies on the induction of tolerance in organ transplantation and experimental autoimmune diseases.

Formation of dichloroacetic acid by rat and mouse gut microflora, an in vitro study.

Metabolism of trichloroethylene (TRI) and its major metabolite, trichloroacetic acid (TCA), by gut content and gut microflora cultures was studied to gain an insight into the role of enterohepatic circulation in TRI metabolism. TRI and TCA were incubated anaerobically with rat and mouse cecal contents and TCA was additionally incubated anaerobically and aerobically with microflora cultures from mice. Although TRI was not metabolized by rat or mouse cecal contents. TCA was metabolized to dichloroacetic acid (DCA) by cecal contents. DCA formation in microflora cultures was dependent on initial TCA concentration, duration of incubation, and initial bacterial number. DCA was not observed in aerobic cultures exposed to TCA. These results suggest that strict anaerobic microorganisms of the gut may partly be responsible for dechlorination of TCA to DCA.

Effects of a growth hormone receptor antagonist on bone markers in acromegaly.

OBJECTIVE: Excess GH secretion, as occurs in acromegaly, is associated with abnormalities in bone turnover markers and bone mineral density (BMD). GH administration in GH deficient patients causes an increase in bone turnover. IGF-I mediates many of the metabolic actions of GH, although GH may have direct effects upon bone. In patients with acromegaly who are treated with a GH receptor antagonist, selective blockade of the GH receptor results in a decrease in circulating IGF-I levels in the majority of cases. We hypothesized that, in acromegaly, antagonism of GH receptors would result in a decrease in serum markers of bone turnover, including serum procollagen I carboxy-terminal propeptide (PICP), osteocalcin and N-telopeptide (NTx). DESIGN AND SUBJECTS: Twenty-seven patients with acromegaly were enrolled as part of a multicentre 12-week trial of a GH receptor antagonist and were randomized to placebo (n = 7) or 10, 15 or 20 mg of pegvisomant (n = 20). MEASUREMENTS: Serum markers of bone turnover were determined at baseline and 12 weeks. RESULTS: Baseline bone turnover markers were above the upper limit of normal in 23%, 19% and 32% of subjects for osteocalcin, PICP and NTx, respectively. During the 12-week placebo-controlled period, there were significant decreases in serum markers of bone formation, osteocalcin (-2.2 +/- 0.44 vs. placebo +0.01 +/- 0.39 nmol/l, P = 0.009) and PICP (-23.6 +/- 9.6 vs. placebo +18.1 +/- 12.8 micro g/l, P = 0.022) and a serum marker of bone resorption, NTx (-4.4 +/- 1.4, placebo +1.0 +/- 0.3 nm, P = 0.024). CONCLUSIONS: Using a specific GH receptor antagonist, we found that normalization of IGF-I is associated with rapid reductions in markers of both bone formation and resorption, and that these processes remain coupled. These data confirm the highly significant effects of GH and IGF-I in modulating bone turnover. The independent contributions of GH and IGF-I to these effects and the long-term effects on BMD in this population remain to be determined.

Depletion of T lymphocytes with immunotoxin retards the progress of experimental allergic encephalomyelitis in rhesus monkeys.

FN18-CRM9 is an anti-rhesus anti-CD3 immunotoxin that can transiently deplete T cells to 1% of initial values in both the blood and lymph node compartments and can induce long-term tolerance to mismatched renal allografts. We have investigated the ability of this immunotoxin to interdict the course of an experimental rhesus T-cell-driven autoimmune disease, experimental allergic encephalomyelitis (EAE) induced by myelin basic protein. Monkeys showing CSF pleocytosis were then treated with FN18-CRM9 alone or in combination with cranial irradiation (325 or 650 cGy). EAE in nontreated control monkeys progressed rapidly. Paralysis occurred 4-6 days after CSF pleocytosis. Paralysis was either delayed or never occurred in treated monkeys, and histopathology revealed few inflammatory plaques that were notable for their low or absent T cell content. While T cells repopulate in the periphery posttreatment, they do not return to the CNS in large numbers, suggesting that the newly repopulated T cells have lost their previously acquired CNS homing capability. Anti-CD3 immunotoxin may be useful in treating clinical T-cell-driven autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Hypogonadism in patients with acromegaly: data from the multi-centre acromegaly registry pilot study.

OBJECTIVE: Because acromegaly is an uncommon disorder, epidemiological data regarding the demographics of the disease such as the prevalence of hypogonadism have been limited. In order to derive clinical and epidemiological information, including underlying hormonal factors, regarding hypogonadism in patients with acromegaly, we performed a pilot study designed to develop a multi-centre acromegaly patient registry. DESIGN AND MEASUREMENTS: Medical records of patients with acromegaly seen between 1976 and 1996 at three Institutions were reviewed, and data were entered into a database using a secure internet website. Hypogonadism was defined as amenorrhoea in women and testosterone deficiency in men. Subanalysis was performed in patients with microadenomas and women less than 50 years of age, to include women of reproductive age. RESULTS: Information was available on 363 patients, of whom 54% were women. The mean age at diagnosis was 41 +/- 13 years. In subjects less than 50 years of age, hypogonadism was present in 59%. Hyperprolactinaemia was present in 45% and 21% of hypogonadal and eugonadal patients of reproductive age, respectively (P = 0.0003). GH levels were higher in patients with hypogonadism (P = 0.03). In patients < 50 years of age with microadenomas, hypogonadism was present in nine of the 22 (41%) patients, including 55% of the women and 27% of the men (P = ns). Hyperprolactinaemia was present in three of the 10 and four of the 14 of microadenoma patients with hypogonadism and eugonadism, respectively. CONCLUSION: We developed a web-based acromegaly patient registry and used it to show that hypogonadism is a frequent consequence of acromegaly, even in patients with microadenomas, who are not at risk from hypopituitarism due to local mass effects. We also demonstrated that prolactin and GH hypersecretion contribute to the pathogenesis of hypogonadism in acromegaly, and that hypogonadism may occur in microadenoma patients even in the absence of hyperprolactinaemia.

Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion.

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.

Long-term treatment of acromegaly with pegvisomant, a growth hormone receptor antagonist.

BACKGROUND: Pegvisomant is a new growth hormone receptor antagonist that improves symptoms and normalises insulin-like growth factor-1 (IGF-1) in a high proportion of patients with acromegaly treated for up to 12 weeks. We assessed the effects of pegvisomant in 160 patients with acromegaly treated for an average of 425 days. METHODS: Treatment efficacy was assessed by measuring changes in tumour volume by magnetic resonance imaging, and serum growth hormone and IGF-1 concentrations in 152 patients who received pegvisomant by daily subcutaneous injection for up to 18 months. The safety analysis included 160 patients some of whom received weekly injections and are excluded from the efficacy analysis. FINDINGS: Mean serum IGF-1 concentrations fell by at least 50%: 467 mg/L (SE 24), 526 mg/L (29), and 523 mg/L (40) in patients treated for 6, 12 and 18 months, respectively (p<0.001), whereas growth hormone increased by 12.5 mg/L (2.1), 12.5 mg/L (3.0), and 14.2 mg/L (5.7) (p<0.001). Of the patients treated for 12 months or more, 87 of 90 (97%) achieved a normal serum IGF-1 concentration. In patients withdrawn from pegvisomant (n=45), serum growth hormone concentrations were 8.0 mg/L (2.5) at baseline, rose to 15.2 mg/L (2.4) on drug, and fell back within 30 days of withdrawal to 8.3 mg/L (2.7). Antibodies to growth hormone were detected in 27 (16.9%) of patients, but no tachyphylaxis was seen. Serum insulin and glucose concentrations were significantly decreased (p<0.05). Two patients experienced progressive growth of their pituitary tumours, and two other patients had increased alanine and asparate aminotransferase concentrations requiring withdrawal from treatment. Mean pituitary tumour volume in 131 patients followed for a mean of 11.46 months (0.70) decreased by 0.033 cm(3) (0.057; p=0.353). INTERPRETATION: Pegvisomant is an effective medical treatment for acromegaly.

Treatment of acromegaly with the growth hormone-receptor antagonist pegvisomant.

BACKGROUND: Patients with acromegaly are currently treated with surgery, radiation therapy, and drugs to reduce hypersecretion of growth hormone, but the treatments may be ineffective and have adverse effects. Pegvisomant is a genetically engineered growth hormone-receptor antagonist that blocks the action of growth hormone. METHODS: We conducted a 12-week, randomized, double-blind study of three daily doses of pegvisomant (10 mg, 15 mg, and 20 mg) and placebo, given subcutaneously, in 112 patients with acromegaly. RESULTS: The mean (+/-SD) serum concentration of insulin-like growth factor I (IGF-I) decreased from base line by 4.0+/-16.8 percent in the placebo group, 26.7+/-27.9 percent in the group that received 10 mg of pegvisomant per day, 50.1+/-26.7 percent in the group that received 15 mg of pegvisomant per day, and 62.5+/-21.3 percent in the group that received 20 mg of pegvisomant per day (P<0.001 for the comparison of each pegvisomant group with placebo), and the concentrations became normal in 10 percent, 54 percent, 81 percent, and 89 percent of patients, respectively (P<0.001 for each comparison with placebo). Among patients treated with 15 mg or 20 mg of pegvisomant per day, there were significant decreases in ring size, soft-tissue swelling, the degree of excessive perspiration, and fatigue. The score fortotal symptoms and signs of acromegaly decreased significantly in all groups receiving pegvisomant (P< or =0.05). The incidence of adverse effects was similar in all groups. CONCLUSIONS: On the basis of these preliminary results, treatment of patients who have acromegaly with a growth hormone-receptor antagonist results in a reduction in serum IGF-I concentrations and in clinical improvement.