The haploinsufficient Col3a1 mouse as a model for vascular Ehlers-Danlos syndrome.

Vascular Ehlers-Danlos syndrome is a rare genetic disorder resulting from mutations in the α-1 chain of type III collagen (COL3A1) and manifesting as tissue fragility with spontaneous rupture of the bowel, gravid uterus, or large or medium arteries. The heterozygous Col3a1 knockout mouse was investigated as a model for this disease. The collagen content in the abdominal aorta of heterozygotes was reduced, and functional testing revealed diminishing wall strength of the aorta in these mice. Colons were grossly and histologically normal, but reduced strength and increased compliance of the wall were found in heterozygotes via pressure testing. Although mice demonstrated no life-threatening clinical signs or gross lesions of vascular subtype Ehlers-Danlos syndrome type IV, thorough histological examination of the aorta of heterozygous mice revealed the presence of a spectrum of lesions similar to those observed in human patients. Lesions increased in number and severity with age (0/5 [0%] in 2-month-old males vs 9/9 [100%] in 14-month-old males, P < .05) and were more common in male than female mice (23/26 [88.5%] vs 14/30 [46.7%] in 9- to 21-month-old animals, P < .05). Haploinsufficiency for Col3a1 in mice recapitulates features of vascular Ehlers-Danlos syndrome in humans and can be used as an experimental model.

A contiguous deletion syndrome of X-linked agammaglobulinemia and sensorineural deafness.

Hearing loss in patients with X-linked agammaglobulinemia is often attributed to recurrent infections. However, recent genetic studies suggest a different etiology in some patients. We present three unrelated patients, 6, 9, and 14 years of age, with large deletions of the terminal portion of the Bruton tyrosine kinase (Btk) gene extending 4.2-19 kb beyond the 3' end of the gene. The DNA immediately downstream of the 3' end of Btk contains the deafness-dystonia protein gene (DDP). Mutations in this gene have recently been shown to underlie the Mohr-Tranebjaerg syndrome, which is characterized by sensorineural deafness, dystonia, and mental deficiency. Besides the immunodeficiency, our patients exhibited progressive sensorineural deafness. The clue to an associated hearing problem was delayed development of speech in one patient and post-lingual deafness noticed between the age of 3-4 years in the other two. These patients have not yet exhibited significant associated neurologic deficits.

Genetic linkage of hyper-IgE syndrome to chromosome 4.

The hyper-IgE syndrome (HIES) is a rare primary immunodeficiency characterized by recurrent skin abscesses, pneumonia, and highly elevated levels of serum IgE. HIES is now recognized as a multisystem disorder, with nonimmunologic abnormalities of the dentition, bones, and connective tissue. HIES can be transmitted as an autosomal dominant trait with variable expressivity. Nineteen kindreds with multiple cases of HIES were scored for clinical and laboratory findings and were genotyped with polymorphic markers in a candidate region on human chromosome 4. Linkage analysis showed a maximum two-point LOD score of 3.61 at recombination fraction of 0 with marker D4S428. Multipoint analysis and simulation testing confirmed that the proximal 4q region contains a disease locus for HIES.

Hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency.

BACKGROUND: Since 1968 it has been known that bone marrow transplantation can ameliorate severe combined immunodeficiency, but data on the long-term efficacy of this treatment are limited. We prospectively studied immunologic function in 89 consecutive infants with severe combined immunodeficiency who received hematopoietic stem-cell transplants at Duke University Medical Center between May 1982 and September 1998. METHODS: Serum immunoglobulin levels and lymphocyte phenotypes and function were assessed and genetic analyses performed according to standard methods. Bone marrow was depleted of T cells by agglutination with soybean lectin and by sheep-erythrocyte rosetting before transplantation. RESULTS: Seventy-seven of the infants received T-cell-depleted, HLA-haploidentical parental marrow, and 12 received HLA-identical marrow from a related donor; 3 of the recipients of haploidentical marrow also received placental-blood transplants from unrelated donors. Except for two patients who received placental blood, none of the recipients received chemotherapy before transplantation or prophylaxis against graft-versus-host disease. Of the 89 infants, 72 (81 percent) were still alive 3 months to 16.5 years after transplantation, including all of the 12 who received HLA-identical marrow, 60 of the 77 (78 percent) who were given haploidentical marrow, and 2 of the 3 (67 percent) who received both haploidentical marrow and placental blood. T-cell function became normal within two weeks after transplantation in the patients who received unfractionated HLA-identical marrow but usually not until three to four months after transplantation in those who received T-cell-depleted marrow. At the time of the most recent evaluation, all but 4 of the 72 survivors had normal T-cell function, and all the T cells in their blood were of donor origin. B-cell function remained abnormal in many of the recipients of haploidentical marrow. In 26 children (5 recipients of HLA-identical marrow and 21 recipients of haploidentical marrow) between 2 percent and 100 percent of B cells were of donor origin. Forty-five of the 72 children were receiving intravenous immune globulin. CONCLUSIONS: Transplantation of marrow from a related donor is a life-saving and life-sustaining treatment for patients with any type of severe combined immunodeficiency, even when there is no HLA-identical donor.

The safety evaluation of fluvastatin, an HMG-CoA reductase inhibitor, in beagle dogs and rhesus monkeys.

Fluvastatin is a potent synthetic competitive inhibitor of beta-hydroxy-beta-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the biosynthetic pathway for hepatic cholesterol synthesis. The therapeutic indication is reduction of elevated total and low-density lipoprotein cholesterol levels. Results from four toxicity studies in beagle dogs and one study in rhesus monkeys following oral administration of fluvastatin are reported. In two 26-week dog studies, doses were 0, 1, 8, or 48 mg/kg/day (reduced to 36 mg/kg/day in Week 7) and 0, 6, 24, or 36 mg/kg/day (reduced to 30 mg/kg/day in Week 2). In a 2-year dog study, doses were 0, 1, 8, or 16 mg/kg/day. Dose levels in the 26-week monkey study were 0, 0.6, 12, and 48 mg/kg/day (raised to 84 mg/kg/day in Week 17 and to 108 mg/kg/day in Week 22). In these studies, evaluations included clinical and physical examinations, body weight and food consumption, electrocardiography, ophthalmoscopy, hematology and clinical chemistries, urinalysis, blood drug concentration, and macroscopic and microscopic examinations of observed lesions and representative tissues. In the 26- and 52-week dog studies and the monkey study, lenticular biochemistry, the HMG-CoA reductase activity of liver microsomes, and serum lipid concentrations were investigated. The fourth dog study was a single-dose toxicokinetic study in which 48 mg/kg [3H]-fluvastatin was monitored for up to 2 weeks. Sampling was limited to ocular tissues for enzyme analysis. Doses of > or = 24 mg/kg/day were lethal in dogs. At lethal doses, ataxia, convulsions, fecal blood, multifocal congestion and hemorrhage, isolated foci of malacia in the medulla oblongata, and liver necrosis were observed. Reduced weight gain, emesis, cataracts, elevated liver enzymes, reduced cholesterol, and gallbladder inflammation with mucosal hyperplasia occurred at > or = 8 mg/kg/day. In contrast to other HMG-CoA reductase inhibitors, fluvastatin did not cause significant central nervous system hemorrhage or testicular changes in dogs. Monkeys tolerated exposure to fluvastatin well with only mild gallbladder changes observed. Reduced serum cholesterol and slight hyperplasia of the gallbladder mucosa occurred in the 12 and 48/84/108 mg/kg/day groups.

The tolerability and pharmacology of interleukin-6 administered in combination with GM-CSF or G-CSF in the rhesus monkey.

The tolerability and potential target organ toxicity of rhIL-6 administered subcutaneously (s.c.) with rhGM-CSF or rhG-CSF were investigated in healthy nonhuman primates. Fifteen Rhesus monkeys were randomized to receive one of the following five regimens: rhIL-6, rhGM-CSF, rhG-CSF, rhIL-6 and rhGM-CSF, or rhIL-6 and rhG-CSF. Each cytokine was administered s.c. once daily at 20 micrograms/kg/day for 30-31 days. Marked increases in blood leukocyte counts (predominantly neutrophils) were observed in the rhGM-CSF and rhG-CSF treatment groups, but only a mild trend toward increased WBCs was observed with rhIL-6 alone. Platelet counts increased 1.7- to 2.2-fold in the rhIL-6 and rhGM-CSF groups. All regimens were well tolerated. RhIL-6, alone or in combination with either CSF, had no significant toxic effects at the dosages tested. Minimal to moderate bone marrow hyperplasia was observed in all except rhIL-6-treated animals, which correlated well with peripheral blood increases in WBCs. RhIL-6-treated animals demonstrated increased fibrinogen concentrations and erythrocyte sedimentation rates, decreased serum albumin/globulin ratios, and increased serum alpha-2-macroglobulin concentrations. Increased synthesis of acute-phase proteins was not observed in the other groups. Combining rhIL-6 with rhGM-CSF or rhG-CSF may reduce the rhIL-6-mediated acute-phase response while maintaining the desirable hematopoietic effects of the stimulating factors.

Pharmacodynamics of daily subcutaneous recombinant human interleukin-3 in normal volunteers.

Normal volunteers received subcutaneous injections of recombinant human interleukin-3 (rhIL-3) on 4 consecutive days to characterize toxicity, pharmacokinetics, and hematopoietic effects. Dosages were 2.5, 5.0, and 7.5 micrograms/kg/day (n = 6 subjects per group). Adverse effects consisted predominantly of flu-like symptoms such as fever and headache. Mean area under the serum concentration-time curve and maximum serum concentration were linearly related to dose. Serum clearance was not apparently related to dose. Clearance increased slightly but significantly between days 1 and 4. Rapid but modest elevations in neutrophil and eosinophil counts were observed during treatment. Mean platelet counts rose modestly, peaking on day 10. Increases of CD34+ cell counts were correlated with increases of colony-forming unit-granulocyte macrophage (peak, day 7).

Therapeutic efficacy of recombinant human leukemia inhibitory factor in a primate model of radiation-induced marrow aplasia.

The therapeutic efficacy of recombinant human leukemia inhibitory factor (LIF) was examined in a nonhuman primate model of radiation-induced marrow aplasia. Rhesus monkeys received 450 cGy of total-body, 1:1 mixed neutron:gamma radiation. For 23 days thereafter, each monkey received a daily subcutaneous injection of LIF or human serum albumin (HSA) at a dose of 15 micrograms/kg body weight. Complete blood counts and white blood cell differentials were monitored for 60 days postirradiation. Administration of LIF significantly decreased (P < or = .05) the duration of thrombocytopenia (platelet count < 30,000 or 20,000/microL), ie, 9.3 days or 6.3 days, respectively, versus the HSA-treated control monkeys, 12.2 days or 10.2 days, respectively. Treatment with LIF did not alter the duration of neutropenia (absolute neutrophil count < 1,000/microL) as compared with the HSA-treated control monkeys. Cytokine administration did not exacerbate the radiation-induced anemia observed in the HSA-treated control monkeys.

Therapeutic efficacy of recombinant interleukin-6 (IL-6) alone and combined with recombinant human IL-3 in a nonhuman primate model of high-dose, sublethal radiation-induced marrow aplasia.

Using a nonhuman-primate model of radiation-induced bone marrow aplasia, we examined whether the single, concomitant, or sequential administration of recombinant human interleukin-3 (IL-3) and IL-6 would promote bone marrow regeneration measured by an increase in circulating platelets (PLT) and neutrophils (PMN). Rhesus monkeys were irradiated at 450 cGy and were randomly assigned to one of five treatment protocols, receiving IL-6; IL-3; combined IL-6 and IL-3; sequential IL-3 and IL-6; or human serum albumin (HSA) as a control. Cytokines or HSA were administered at total dosages of 15 micrograms/kg/day. Complete blood counts and white blood cell differentials were monitored for 60 days postirradiation. Both IL-3 and IL-6 significantly enhanced the regeneration of PLTs and decreased the duration of thrombocytopenia (P = .005) without affecting PMN recovery. The radiation-induced anemia that was observed in the HSA-treated controls was less severe and resolved more quickly in the IL-6 treated animals. Sequential IL-3/IL-6 significantly increased the production of PLTs when compared with the HSA-treated controls (P = .003) and monkeys receiving concomitant IL-3/IL-6 (P = .041) but did not alter PMN levels significantly (P = .80). Coadministration of IL-6 and IL-3 did not enhance PLT but improved PMN recovery over IL-6 alone. In this primate model of marrow aplasia, IL-6 significantly enhanced the regeneration of PLTs but had no significant effect on PMN production, and did not exacerbate radiation-induced anemia. Furthermore, the use of sequentially administered IL-3 and IL-6 may improve PLT recovery as compared with concurrent IL-3/IL-6 administration, although this protocol is not significantly different in effect than either cytokine alone.

Effect of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on hematopoietic regeneration as demonstrated in a nonhuman primate chemotherapy model.

Using a recently developed hepsulfam-induced pancytopenia model in rhesus macaques, we have studied the effects of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on marrow regeneration. Control animals were given hepsulfam (1.5 g/m2 by a single 30-minute intravenous [i.v.] injection, n = 4), while study animals received hepsulfam followed by rhIL-6, rhIL-3, or a combination of rhIL-6 and rhIL-3 (n = 3 per study group). Each cytokine was administered by once-daily subcutaneous (SC) injection (15 micrograms/kg/d) for 3 weeks beginning the day after chemotherapy (days 2 through 22). Mean platelet counts in control animals were < 100,000/microL on days 15 through 24, with 50% of the counts < 50,000/microL and two of four animals requiring platelet transfusion. In the rhIL-6- and rhIL-6/rhIL-3-treated groups, the nadir mean platelet counts were 164,000 +/- 58,700/microL and 162,300 +/- 23,800/microL, respectively, and occurred on day 15. Platelet counts in the rhIL-3-treated group were similar to those in controls. Mean absolute neutrophil counts (ANCs) < 1,000/microL occurred on days 10 through 29 in control animals, days 8 through 15 in rhIL-6-treated animals, and days 6 through 8 and 13 in rhIL-6/rhIL-3-treated animals. The frequency of ANCs < 500/microL was significantly less in the rhIL-6- and rhIL-6/rhIL-3-treated groups versus control groups (2.7 +/- 0.6 and 2.0 +/- 1.0 vs 7.0 +/- 1.4 occurrences, respectively; P < .05). rhIL-3-treated animals had ANCs similar to those in controls; one animal died with septicemia on day 21. Monkeys receiving rhIL-6 were significantly more anemic during the cytokine administration period; however, the anemia resolved by day 24. Coadministration of rhIL-3 and rhIL-6 partially corrected the anemia. The data indicate that rhIL-6 prevents significant thrombocytopenia and shortens the neutropenic period in this chemotherapy model.

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on platelet survival and activation using a nonhuman primate model.

In humans and nonhuman primates, the in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) consistently results in marked increase of megakaryocyte ploidy and size similar to that observed with interleukin-6 (IL-6). However, whereas the administration of IL-6 also results in an increase in circulating platelets, there is no predictable corresponding increase in peripheral blood platelets following treatment with rhGM-CSF. To determine whether the failure of rhGM-CSF to produce thrombocytosis is secondary to cytokine-related increased platelet activation and consumption in vivo, we quantified autologous platelet survival time and in vivo platelet activation before and during 5 days of administration of rhGM-CSF to two rhesus monkeys. Platelet survival was measured using autologous platelets labeled with 111Indium-oxine. Platelet activation was assessed by flow cytometric determination of the expression of the major platelet membrane glycoprotein (GP) IIb/IIIa complex, and an activation-dependent epitope on GPIIb/IIIa (recognized by monoclonal antibodies [MABs] LJ-P4 and PAC1, respectively). Platelet activation was also assessed by dose-response aggregometry using adenosine diphosphate (ADP). While megakaryocyte ploidy increased during rhGM-CSF administration, peripheral platelet counts were 418 x 10(9)/L and 525 x 10(9)/L before and 402 x 10(9)/L and 508 x 10(9)/L during cytokine treatment in animals 1 and 2, respectively. No changes were observed in the mean platelet volume. 111Indium-labeled platelet recovery in circulation was similar before (94.7%, 91.8%) and during (92.9%, 92.8%) rhGM-CSF administration, which indicates that cytokine-related in vivo sequestration of platelets does not occur. Autologous platelet survival was 5.6 and 6.2 days before and 5.0 and 5.4 days during the rhGM-CSF treatment (p = 0.07), without significant change in the corresponding platelet turnover rate (derived from the platelet count and survival time). The flow cytometric analysis showed no increase in the binding of either LJ-P4 or PAC1 MABs to the platelet membrane during rhGM-CSF administration. The aggregometry studies demonstrated similar concentrations of ADP inducing half-maximal aggregation (ED50). Overall, the above data indicate that treatment with rhGM-CSF is not associated with in vivo activation, sequestration, or increased consumption of platelets. The data suggest that the failure of rhGM-CSF-stimulated megakaryocytes to increase peripheral platelet count is a manifestation of ineffective megakaryocytopoiesis resulting from inability to increase platelet delivery to the circulation.

Studies of an improved rhesus hematopoietic progenitor cell assay.

We have improved Rhesus monkey marrow cell growth in semisolid media by means of substituting supplemented calf serum for fetal bovine serum. The cloning efficiency of light-density marrow cells separated on 60% Percoll was 126 (+/- 54)/10(5) (n = 12, +/- SD), and for light-density peripheral blood cells 60 (+/- 46)/10(6) (n = 11). Thirty-five percent of the colonies were multilineage, whereas the remainder were unilineage colonies composed of erythrocytes, megakaryocytes, and neutrophilic or monocytic granulocytes. Unilineage megakaryocyte colonies comprised 12% of the total marrow progenitor cells. The [3H]TdR suicide index of marrow progenitor cells was 47% +/- 9% (n = 12). This progenitor cell assay should prove useful in preclinical studies of the effect of recombinant hematopoietic growth factors on the number and cycling status of Rhesus hematopoietic progenitor cells.

Recombinant human granulocyte-macrophage colony-stimulating factor promotes megakaryocyte maturation in nonhuman primates.

Megakaryocytes are responsive to several nonlineage-specific cytokines in vitro. In this study, we examined the in vivo effects of recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) on late stages of megakaryocytopoiesis in the rhesus monkey. Four rhesus monkeys were given 10 micrograms/kg body weight/day of rh GM-CSF s.c. in two divided doses daily for 8 days. Megakaryocyte maturation was evaluated serially by measuring nuclear ploidy and cytoplasmic size. GM-CSF-treated monkeys developed significant shifts in ploidy distribution from days 3 through 15 (p less than or equal to 0.001), with increased frequencies of 64N and 128N megakaryocytes. Mean megakaryocyte size increased 92.5% on day 9, paralleling the increase in DNA content, although megakaryocyte size within ploidy groups did not increase. Megakaryocyte number remained unchanged following rh GM-CSF treatment. The platelet count responses were variable, and mean platelet volume did not change. The present study demonstrates that therapeutic doses of rh GM-CSF stimulate megakaryocyte endomitosis and increase mean size. The data indicate that GM-CSF is effective in promoting the maturation stage of megakaryocyte development but does not result in a consistent thrombopoietic response.

Characterization and identification of an indirect cytochrome P-450-initiated denitrosation of 2,6-dichloro-4-nitroaniline in rat hepatic microsomes.

The metabolism of 2,6-dichloro-4-nitroaniline (DCNA) to a unique denitrosated product, 3,5-dichloro-p-aminophenol (DCAP), was investigated in rat hepatic microsomes using an HPLC system containing a reverse-phase column and an electrochemical detector. The parent compound appears to induce its own metabolism. The characterization of this induction was studied by polyacrylamide gel electrophoresis, catalytic enzymatic activity, and immunochemistry. The in vitro microsomal aerobic production of DCAP was increased 4- to 6.5-fold with respect to controls after animals were treated with DCNA. The microsomal production of DCAP can be inhibited by the addition of specific antibodies to cytochrome P-450d, thus indicating that the removal of the nitro group and subsequent replacement with a hydroxyl group was initiated by cytochrome P-450d in the mixed-function oxidase system. Finally, it was demonstrated by the addition of H218O to the assay that this hydroxyl group came from H2O and not molecular oxygen. It is concluded that cytochrome P-450 initiated this novel reaction by the formation of an N-hydroxylamine, followed by a non-P-450-mediated attack of water causing the removal of nitrous acid and the formation of the phenol.