Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa

Controlling the sex of offspring by the separation of X and Y chromosome-bearing spermatozoa using flow cytometry has been reported as a clinical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic safety due to the involvement of two potentially mutagenic agents: UV light and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with other mammalian species. The stability of normal fresh and decondensed human spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh and frozen-thawed human spermatozoa was examined following UV laser, H33342 staining and flow cytometry treatments utilizing the same assay. There was an increase in the presence of endogenous nicks when spermatozoa were decondensed compared with fresh spermatozoa. There was no increase in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.      

Effects of monoclonal antibody therapy in patients with chronic lymphocytic leukemia

A phase I clinical trial was initiated to treat patients with stage IV B-derived chronic lymphocytic leukemia (CLL) with the IgG2a murine monoclonal antibody T101. This antibody binds to a 65,000-mol wt (T65) antigen found on normal T lymphocytes, malignant T lymphocytes, and B- derived CLL cells. All of the patients had a histologically confirmed diagnosis of advanced B-derived CLL and were refractory to standard therapy, and more than 50% of their leukemia cells reacted with the T101 antibody in vitro. The patients received T101 antibody two times per week, over two to 50 hours by intravenous administration in 100 mL of normal saline containing 5% human albumin. Twelve patients were treated with a fixed dosage of 1, 10, 50, or 100 mg, and one patient was treated with 140 mg of antibody. It was demonstrated that patients given two-hour infusions of 50 mg developed pulmonary toxicity, with shortness of breath and chest tightness. This toxicity was eliminated when infusions of 50 or 100 mg of T101 were prolonged to 50 hours. All dose levels caused a rapid but transient decrease in circulating leukemia cell counts. In vivo binding to circulating and bone marrow leukemia cells was demonstrated at all dose levels with increased binding at higher dosages. Antimurine antibody responses were not demonstrated in any patients at any time during treatment. Circulating free murine antibody was demonstrated in the serum of only the two patients treated with 100 mg of antibody as a 50-hour infusion and the patient treated with 140 mg of antibody over 30 hours. Antigenic modulation was demonstrated in patients treated at all dose levels but was particularly apparent in patients treated with prolonged infusions of 50 and 100 mg of antibody. We were also able to demonstrate antigenic modulation in lymph node cells, which strongly suggests in vivo labeling of these cells. Overall, T101 antibody alone appears to have a very limited therapeutic value for patients with CLL. The observations of in vivo labeling of tumor cells, antigenic modulation, antibody pharmacokinetics, toxicity, and antimurine antibody formation may be used in the future for more effective therapy when drugs or toxins are conjugated to the antibody.     

Existence of a pool of T-lymphocyte colony-forming cells (T-CFC) in human bone marrow and their place in the differentiation of the T- lymphocyte lineage

The existence and characteristics of bone marrow T-cell progenitors have not yet been established in man. Several pieces of evidence such as the reconstitution of certain immunodeficiencies by bone marrow graft suggest that T-cell precursors are present in the bone marrow. We report the growth of T-cell colonies from bone marrow populations using PHA-stimulated lymphocyte-conditioned medium containing T-cell growth factor (TCGF). Rosetting experiments and complement-dependent cytotoxicity assays with monoclonal antibodies indicate that the bone marrow T colony-forming cells (T-CFC) are E- OKT 3- and la+, i.e., immature progenitors. The colonies derived from these cells have the phenotype of mature T cells: E + OKT 3 + la- with either helper (OKT 4+) and suppressor (OKT 8 +) antigens. These results suggest that a thymic microenvironment may not be necessary for the in vitro proliferation and differentiation of the T-cell lineage in adult humans. These methodologies may permit direct investigation of early phenomena concerning the T-cell lineage, such as the acquisition of self-tolerance, the formation of a repertoire of specificities, and the HLA restriction phenomena that we believe takes place before the thymic maturation.     

Nasal inflammation and chronic ear disease in Australian Aboriginal children

Objective : Chronic middle ear disease is common in Aboriginal children, and may be linked to nasal inflammation and Eustachian tube dysfunction. The pattern of nasal inflammation is unknown. The study reported here was performed to define the role of allergy and infection in causing nasal inflammation in Aboriginal children with chronic middle ear disease.
Methodology : Thirty-one Aboriginal children aged between 3 and 7 years underwent clinical assessment, audiometry and allergy skin tests. Nasal swabs for bacterial culture and cytology were performed during the winter and again in spring to identify any seasonal variation. A randomized trial of nasal beclomethasone for 8 weeks was conducted in children with abnormal tympanometry to identify the effect of therapy upon nasal cytology.
Results : Twenty-six of the 31 children had abnormal tympanograms. Average hearing levels were reduced in nine children. Pathogenic organisms were isolated from most children: Streptococcus pneumoniae (82%), Haemophilus influenzae (79%), Moraxella catarrhalis (39%) and Staphylococcus aureus (29%). Eight of the 31 children (26%) were atopic. Nasal cytology disclosed a marked neutrophil infiltrate (80% of cells) during the winter, which fell significantly in spring to 52% of cells. Only two subjects had nasal eosinophilia of >10%. There was no effect of beclomethasone on nasal cytology.
Conclusions : Chronic ear disease in Aboriginal children is associated with nasal inflammation, neutrophil infiltration and the presence of bacteria. These features suggest respiratory infection as the main cause of chronic nasal inflammation in Aboriginal children with middle ear disease. There is a seasonal variation in the severity of the nasal infiltrate, consistent with increased infections during winter. Despite a high prevalence of atopy, allergic nasal disease was uncommon.     

Pericardial effusion complicating a percutaneous central venous line in a neonate

A premature infant developed pericardial effusion four days after the insertion of a 25-gauge silastic percutaneous central venous catheter. The effusion contained parenteral nutrition fluid and resolved rapidly after withdrawal of the catheter. Pericardial effusion is a potential complication of percutaneous, as well as surgically placed, central venous catheters.     

Mental health and psychosocial functioning in adolescents with congenital heart disease. A comparison between adolescents born with severe heart defect and atrial septal defect

Twenty-six adolescents, aged 13–18 years, with severe congenital heart disease were matched for sex, age and living area with 26 adolescents with repaired atrial septal defect and regarded as physically fit. These two groups were compared according to somatic condition, psychopathology, psychosocial functioning and chronic family difficulties. A higher rate of psychiatric problems in the complex group, an association between psychosocial functioning and physical capacity, as well as an association between psychosocial functioning and chronic family difficulties were observed. These findings suggest that physical capacity is of crucial importance for mental health and functioning of adolescents with congenital heart disease. The association with chronic family difficulties also suggests that a comprehensive biopsychosocial approach is necessary in the treatment and rehabilitation of these patients.     

Auxology and response to growth hormone treatment of patients with intrauterine growth retardation or Silver-Russell syndrome: analysis of data from the Kabi Pharmacia International Growth Study

Using the database from the Kabi Pharmacia International Growth Study, 105 patients with intrauterine growth retardation (IUGR) (82 males, 23 females) and 45 with Silver-Russell syndrome (SRS) (32 males, 13 females) with persistent postnatal growth failure were studied. Patients with IUGR had a birth length and birth weight more than 2 SD below the mean for gestational age. Their height deficit at the start of GH treatment was -3.0 SDS at a median chronological age of 8.7 years and a median bone age of 7.0 years. Mean paternal and maternal heights were 166 and 153 cm, respectively. The median dose of GH was 0.5 IU/kg/week, given at a median frequency of five injections/week. The median height SDS for chronological age after 1, 2 and 3 years of GH treatment were -2.5, -2.1 and -1.9, respectively. In the 45 patients with SRS, median chronological age and median bone age at the start of treatment were 6.7 and 3.2 years, respectively, and mean paternal and maternal heights were 167.5 and 160 cm, respectively. The median dose of GH was 0.7 IU/kg/week, given at a median frequency of six injections/week. The median height SDS for chronological age at the start of treatment and after 1, 2 and 3 years were -3.5, -2.9, -2.8 and -2.2, respectively. Although the criteria used by physicians when diagnosing SRS were not controlled or verified in this study, it appears that patients with SRS can be differentiated from those with IUGR with persistent growth failure by their reduced bone age for chronological age at the start of treatment, and by the fact that patients with SRS tended to be born to parents of normal height. GH treatment in both groups induced catch-up growth, though long-term follow-up studies will be required to assess the effects of treatment on final height.