Late onset of severe graft-versus-host disease in a pediatric liver transplant recipient.

We report the management of a patient with the late onset of chronic graft-versus-host disease (GVHD) after orthotopic liver transplantation. GVHD is a rare complication of solid organ transplants that usually presents early after transplantation and is fatal in the majority of cases. Our patient differs from the typical patient with GVHD in that the onset of her disease was very late. Although most treatment to date consisted of an increase in immunosuppressive therapy, our patient showed an excellent response to a reduction. This resulted in the abatement of the symptoms of GVHD and the preservation of her allograft function.

X microchromosome with additional chromosome anomalies found in Ullrich-Turner syndrome.

Using standard cytogenetic methods coupled with molecular techniques, the following karyotype mos 45,X/46,XXq+/46,X+mar (X)/47,XXq+,+mar(X), was identified in a patient with Ullrich-Turner syndrome (UTS). High-resolution banding (n = 650) of the metaphase chromosomes yielded a breakpoint at q28 on the Xq+ rearranged chromosome. FISH was used to determine the presence of Y-containing DNA in the Xq+ and the mar(X) chromosomes. The following molecular probes were used: DYZ1, DYZ3, and spectrum orange WCP Y. The lack of specific hybridization of these probes was interpreted as a low risk of gonadoblastoma in this patient. Using X-chromosome- and centromere-specific probes, FISH demonstrated the presence of hybridizing material on both rearranged chromosomes, the Xq+ and mar(X). Finally, we determined that the mar(X) and Xq+ chromosomes contained telomeres in the absence of any interstitial telomeric hybridizing material. A micro-X chromosome is present in this UTS patient. Delineation of events leading toward the mechanisms responsible for the multiple DNA rearrangements required to generate the micro-X and Xq+ chromosomes awaits future studies.

Cystic fibrosis-related diabetes is associated with HLA DQB1 alleles encoding Asp-57- molecules.

The incidence of insulin-dependent diabetes in individuals with cystic fibrosis is nearly 100 times greater than in the general population. In the latter group, strong associations with specific HLA DQA1 and DQB1 alleles have been observed. To determine if a similar distribution of alleles occurs in cystic fibrosis patients with diabetes, a cohort of these individuals was typed for DQA1 and DQB1 alleles. HLA DQB1*0201 (Asp57-) was more frequent in diabetics compared to controls (40.4 vs 28%), while the frequency of alleles encoding Asp57+ molecules was lower in diabetics relative to both the cystic fibrosis-only controls (P = 0.025) and the general population (P = 0.008). The presence of at least one protective DQA1-DQB1 heterodimer (i.e., Arg52- and Asp57+, respectively) in cis or trans was significantly lower in the diabetics than in either of the control groups. Thus, the HLA alleles known to be associated with insulin-dependent diabetes mellitus in the general population are also found in diabetics with cystic fibrosis.

Detection of three rare frameshift mutations in the cystic fibrosis gene in an African-American (CF444delA), an Italian (CF2522insC), and a Soviet (CF3821delT).

We have identified three new frameshift mutations in the CFTR gene in patients with cystic fibrosis (CF). The first one involves the deletion of an adenine nucleotide in exon 4 in an African-American patient (CF444delA), the second involves the insertion of a cytosine nucleotide in exon 13 in an Italian patient (CF2522insC), and the third results from the deletion of a thymidine nucleotide in exon 19 in a Soviet patient (CF3821delT). Each mutation is predicted to result in premature termination of the CFTR protein.

Fatty acid uptake and catecholamine-stimulated phospholipid metabolism in immortalized airway epithelial cells established from primary cultures.

The incorporation of [14C]oleic and [14C]linoleic acid into phospholipids and neutral lipids was compared in two recently immortalized airway epithelial cell lines. In addition, the effects of adrenergic stimulation on phospholipid turnover was examined. Both cell lines readily incorporated the fatty acids into all phospholipid and neutral lipid fractions. Isoproterenol (1 microM) induced Ca2+ transients in both cell lines, indicating a functional beta-adrenergic response. Epinephrine (10 microM; 15 min) stimulation of cells prelabeled with [14C]linoleic acid increased the percentage of label in phosphatidylcholine in one cell line. Lipid metabolism can now be extensively studied in human airway epithelia.

Microscopic nephrocalcinosis in cystic fibrosis.

Airway, sweat-duct, and other epithelial cells in patients with cystic fibrosis display abnormal ion transport. To test whether the kidney, the organ most exquisitely adapted for ion transport, has a similar defect, we measured the levels of calcium excretion and searched for microscopic nephrocalcinosis in patients with cystic fibrosis. Thirty-eight specimens of kidney tissue were stained for calcium deposits, and 24-hour levels of urinary calcium excretion were measured in 14 patients and 15 control subjects. Microscopic nephrocalcinosis was observed in 35 of the 38 specimens (92 percent), and hypercalciuria (greater than 182 mg per gram of creatinine) in 5 of the 14 patients (36 percent). Notably, nephrocalcinosis was detected near the time of birth (in six patients under one year old, including two neonates and one stillborn infant), which supports the hypothesis that such renal calcium deposits reflect the genomic defect and are not due to longstanding pulmonary dysfunction, chronic infection, therapeutic agents, or disease progression. None of the patients with hypercalciuria or nephrocalcinosis had clinical evidence of renal dysfunction. The finding of microscopic nephrocalcinosis near the time of birth in patients with cystic fibrosis suggests a primary abnormality of calcium metabolism in the kidney. Studies of the pathophysiologic features of the kidney in cystic fibrosis may elucidate the molecular alterations observed in this disorder.

Nucleic acid hybridization in plasma: method for the quantitation of poly(I).poly(C12, U) in plasma of cancer patients.

Using a new method for the direct measurement of the double-stranded RNA (dsRNA) molecule poly(I).poly(C12, U) in plasma, levels of 100 X 10(-9) g of drug were routinely quantified. The samples were digested by proteinase K in a buffered solution containing 0.1% of Brij-35 and deoxycholate detergents. The digestions were terminated after 1 h by the addition of Brij-58 and boiling saturated NaI (1.67 g/ml). Serially diluted samples were filtered onto nitrocellulose and the filters washed and hybridized. Levels of the hybridized-radioactive probe, synthesized de novo in an RNA dependent DNA transcription system, were determined by liquid scintillation spectrophotometry and quantified by comparison to a standard curve. The efficiency of hybridization declined when the plasma concentration in the reaction fell below 1.0 mg/ml. Incubation and denaturation temperatures significantly altered the amount of radioactive probe hybridized; results varied in the extent of hybridization and in the concentration range of dsRNA showing a linear response. Elevated temperature during proteinase K digestion showed reduced hybridization efficiencies: 100% at 25, 80% at 37, 35% at 45, and 25% at 55 degrees C. Incubation at elevated temperatures, prior to the addition of NaI, caused a decline in the amount of radioactivity hybridized, but did not have an effect during hybridization.

Comparative studies of ampligen (mismatched double-stranded RNA) and interferons.

Historically, it has been assumed that double-stranded (ds) RNAs function at a cellular level exclusively via an interferon (IFN) induction mechanism. However, current studies conducted both in the laboratory and at the clinical level reveal that this assumption is incorrect and, indeed, underestimates the intrinsic antitumor activity of certain dsRNAs. A specific dsRNA (Ampligen) shows strong antiproliferative activity against human carcinoid tumor cells in a clonogenic assay when natural alpha- and beta-IFNs were inactive. Similarly, in vivo studies in which human renal cancer cells were transplanted into athymic mice demonstrate a strong antitumor effect of Ampligen whereas such tumors are largely unaffected by alpha-IFN treatment. In a comparative study including many fresh human tumors of various histological types (breast, ovarian, melanoma, renal, and carcinoid) numerous examples were uncovered of Ampligen sensitivity (antiproliferative effect) in the face of relative or complete insensitivity to IFNs. Synergistic effects of Ampligen plus IFN overcame the resistance of some human tumor cells to either biological modifier given alone. It can also be demonstrated that the antitumor action of Ampligen on certain human lung tumor cells is not shared by polyinosinic . polycytidylic acid, thus indicating that different dsRNAs may themselves exhibit dissimilar effects on various human tumors.

Clinical studies with ampligen (mismatched double-stranded RNA).

Results of an ongoing clinical study of a mismatched double-stranded (ds) RNA, termed Ampligen, in patients with metastatic cancer are described. In a pilot study of Ampligen (lot 1) involving mostly hematologic malignancies, patients received cumulative doses up to approximately 450 mg without untoward effects. Evidence of biologic/antitumor effects was observed (3/5 patients) by monitoring tumor-specific markers or tumor cell morphology. In patients with solid tumors receiving lot 2, Ampligen cumulative doses over 4 g were well tolerated. The drug was given by intravenous infusion (10-80 mg/infusion, twice weekly), in some instances for more than 1 year, without clinically significant side effects. Specifically, no evidence of hematologic, liver, or renal toxicity, which was previously noted with other dsRNAs, was observed. Side effects consisted of occasional mild fatigue or flu-like symptoms. Fever, when encountered, was transient and low grade (less than 100.5 degrees F). Importantly, an analysis of patient sera for dsRNA antibodies revealed that no patient had evidence of specific antibodies directed against Ampligen. Other dsRNAs cause up to a 60% incidence of antibody formation. Additionally, a novel method was developed to monitor Ampligen blood levels. In a survey of seven patients, Ampligen had a mean plasma half-life of 23 min. Ampligen administration can also result in activation of both natural killer (NK) cells and a lymphocyte, interferon-associated, intracellular enzyme. Dose-dependent antitumor effects were seen in several solid tumors; in doses of 10-40 mg, 3/9 patients showed stable disease for up to 1 year. At the 80-mg dose level, 2/5 patients showed tumor regressions (mixed and partial responses).(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-induced growth modulation: low dose maintenance of the antiproliferative response.

Treatment of the Burkitt's lymphoma-derived Daudi cell line with human beta interferon (HuIFN-beta) results in a dose-dependent antiproliferative response. We have defined three phases including: (1) initiation, (2) maintenance, and (3) termination of the antiproliferative state. Each phase is characterized by specific growth modulatory properties. Initiation of the antiproliferative state with a single interferon dose requires 200 International Reference Units (IRU) per ml, depending on such factors including cell density, serum content in the medium and the length of IFN exposure. Initiation of the IFN response occurred within a 4 h incubation period. Even following this short exposure, the vast majority of cells would be sequestered in the G0/G1 cell cycle compartment. As measured by cytofluorimetry there was a 16-20 h delay in the initiation of the antiproliferative response. The antiproliferative response, as measured by changes in the distribution of cells in the cell cycle, was maximal at 24 h after treatment. This delay was equal to the doubling time of the Daudi cell. The antiproliferative response initiated by 200 IRU/ml of IFN was reversible if the IFN was removed and not replenished. Cells showed renewed growth approximately 40 h after treatment. The antiproliferative state could be maintained by additional interferon, but, even after such treatment, the antiproliferative state decayed in a dose-dependent fashion. This decay was proportional to the decrease in the biological activity of the interferon molecule itself. Finally, we showed that the antiproliferative state could be maintained by using only 40 IRU/ml. These cells were maintained in the antiproliferative state for an extended period of time.

Indications of congenital palatal incompetence before diagnosis.

By questionnaire, parents of 28 children with CPI were asked about pregnancy and delivery; speech, developmental, and health history; and the circumstances of the CPI diagnosis. A normal control group was used for comparison. CPI children had lower birth weights and, during early childhood, were more poorly understood by parents and more frequently had nasalized speech. In 50%, CPI diagnosis was made following adenoidectomy. Data and clinical findings indicate that possible predictors of CPI are unusually defective speech production, particularly characterized by nasalization, as a young child; a short or poorly mobile palate; anterior dimpling of the soft palate during elevation; radiographic abnormalities of the cervical vertebrae; neurologic abnormalities (developmental milestones or clinical examination); and nasal leakage of liquids as a very young infant. Temporary velopharyngeal incompetence and nasalized speech may occur in the normal patient following adenoidectomy but the disorder resolves in a day or two. Persistent nasalized speech following adenoidectomy indicates the likelihood of CPI; such a patient requires evaluation by a speech pathologist and possible surgical correction.

Transcription of single base oligonucleotides by ribonucleic acid-directed deoxyribonucleic acid polymerase.

The synthesis of DNA products complementary to artificial templates by the enzyme RNA-directed DNA polymerase isolated from avian myeloblastosis virus has been studied. Of the single base polyribonucleotides, poly (rC), poly(rA), and poly(rI) were active while poly (rG) and poly (rU) were almost inactive. The minimum length showing activity for an oligo (rC) template was 9; the minimum primer length of oligo(dG) was 3 or 4. In order to examine the fidelity of transcription, single base oligoribonucleotides of defined length were studied. Using (rC)13 as template and (dG)8as primer, the oligo (dG) product coelectrophoresed with the template. However, using (rA)-20 as template and (dT)10 as primer, a large (10-16s) product was formed. Similarly, using oligo (rI) (2.5S) as template and (dC)10 as primer, a large (greater than 22s) product was formed. No significant activity was obtained with oligo (rU) templates. RNA-directed DNA polymerase transcribes the various oligonucleotides differently: slippage with oligo (rA) and oligo (rI), faithful transcription with oligo (rC), and poor transcription with oligo (rU).