Effect of cancer chemotherapy on the frequency of minisatellite repeat number changes in human sperm

The objective of this study was to determine whether cancer chemotherapy induces detectable mutations in DNA of the human germline and whether minisatellite repeat number changes can be used as a sensitive indicator of genetic damage in human sperm caused by mutagens. We compared the mutation frequencies in sperm of the same cancer patients pre- and post-, pre- and during, or during and post-treatment. Small pool polymerase chain reaction (SP-PCR) (DNA equivalent to approximately 100 sperm) and Southern blotting techniques were used to detect mutations and quantify the frequency of repeat number changes at the minisatellite MS205 locus. One pre- and one post-treatment semen sample was obtained from each Hodgkin's disease patient treated with either: (1) a regimen without alkylating agents, Novantrone, Oncovin, Vinblastine, and Prednisone (NOVP), 4 patients; (2) a regimen containing alkylating agents, Cytoxan, Vinblastine, Procarbazine, and Prednisone (CVPP)/Adriamycin, Bleomycin, DTIC, CCNU, and Prednisone (ABDIC), 2 patients; and (3) a regimen containing alkylating agents, Mechlorethamine, Oncovin, Procarbazine, and Prednisone (MOPP), 1 patient. One pre- and one during treatment semen sample from each of two Hodgkin's disease patients treated with Adriamycin, Bleomycin, Vinblastine, and Dacarbazine (ABVD) were obtained. One during and one post-treatment semen sample from a Hodgkin's disease patient treated with NOVP were also obtained. At least 7900 sperm in each sample were screened for the repeat number changes at the MS205 locus by multi-aliquots of SP-PCR. The mutation frequencies of pre- and post-treatment for the four patients treated with NOVP were 0.22 and 0.18%; 0.24 and 0.16%; 0.35 and 0.28%; and 0.19 and 0.18%. With CVPP/ABDIC, they were 0.22 and 0.23%; and 0.94 and 0.98% for the two patients and with MOPP they were 0.79 and 1.14%. The mutation frequencies of pre- and during treatment with ABVD were 0.09 and 0.07%; and 0.34 and 0.27% for the two patients. The mutation frequencies of during and post-treatment with NOVP for one patient were 0.31 and 0.25%. A statistically significant increase in mutation frequency was only found in the patient treated with MOPP. According to the time of samples collected after or during treatment and the above results, we conclude that there is no effect of NOVP and CVPP/ABDIC regimens on the mutation frequency in spermatogonia. The spermatocytes are not highly sensitive to chemotherapy agents compared to spermatogonia at the minisatellite MS205 locus. MOPP treatment may increase the mutation frequency at the MS205 locus in spermatogonia. ^

New methods for quantification and analysis of quantitative real-time polymerase chain reaction data

Quantitative real-time polymerase chain reaction (qPCR) is a sensitive gene quantitation method that has been widely used in the biological and biomedical fields. The currently used methods for PCR data analysis, including the threshold cycle (CT) method, linear and non-linear model fitting methods, all require subtracting background fluorescence. However, the removal of background fluorescence is usually inaccurate, and therefore can distort results. Here, we propose a new method, the taking-difference linear regression method, to overcome this limitation. Briefly, for each two consecutive PCR cycles, we subtracted the fluorescence in the former cycle from that in the later cycle, transforming the n cycle raw data into n-1 cycle data. Then linear regression was applied to the natural logarithm of the transformed data. Finally, amplification efficiencies and the initial DNA molecular numbers were calculated for each PCR run. To evaluate this new method, we compared it in terms of accuracy and precision with the original linear regression method with three background corrections, being the mean of cycles 1-3, the mean of cycles 3-7, and the minimum. Three criteria, including threshold identification, max R2, and max slope, were employed to search for target data points. Considering that PCR data are time series data, we also applied linear mixed models. Collectively, when the threshold identification criterion was applied and when the linear mixed model was adopted, the taking-difference linear regression method was superior as it gave an accurate estimation of initial DNA amount and a reasonable estimation of PCR amplification efficiencies. When the criteria of max R2 and max slope were used, the original linear regression method gave an accurate estimation of initial DNA amount. Overall, the taking-difference linear regression method avoids the error in subtracting an unknown background and thus it is theoretically more accurate and reliable. This method is easy to perform and the taking-difference strategy can be extended to all current methods for qPCR data analysis.^

Positional cloning and characterization of the human aniridia and murine small eye genes

Aniridia (AN) is a congenital, panocular disorder of the eye characterized by the complete or partial absence of the iris. The disease can occur in both the sporadic and familial forms which, in the latter case, is inherited as an autosomal dominant trait with high penetrance. The objective of this study was to isolate and characterize the genes involved in AN and Sey, and thereby to gain a better understanding of the molecular basis of the two disorders.^ Using a positional cloning strategy, I have approached and cloned from the AN locus in human chromosomal band 11p13 a cDNA that is deleted in two patients with AN. The deletions in these patients overlap by about 70 kb and encompass the 3$\sp\prime$ end of the cDNA. This cDNA detects a 2.7 kb mRNA encoded by a transcription unit estimated to span approximately 50 kb of genomic DNA. The message is specifically expressed in all tissues affected in all forms of AN, namely within the presumptive iris, lens, neuroretina, the superficial layers of the cornea, the olfactory bulbs, and the cerebellum. Sequence analysis of the AN cDNA revealed a number of motifs characteristic of certain transcription factors. Chief among these are the presence of the paired domain, the homeodomain, and a carboxy-terminal domain rich in serine, threonine and proline residues. The overall structure shows high homology to the Drosophila segmentation gene paired and members of the murine Pax family of developmental control genes.^ Utilizing a conserved human genomic DNA sequence as probe, I was able to isolate an embryonic murine cDNA which is over 92% homologous in nucleotide sequence and virtually identical at the amino acid level to the human AN cDNA. The expression pattern of the murine gene is the same as that in man, supporting the conclusion that it probably corresponds to the Sey gene. Its specific expression in the neuroectodermal component of the eye, in glioblastomas, but not in the neural crest-derived PC12 pheochromocytoma cell line, suggests that a defect in neuroectodermal rather mesodermal development might be the common etiological factor underlying AN and Sey. ^

Use of a hypomorphic allele of myogenin to analyze Myogenin-dependent processes in mouse development

Myogenin is a muscle-specific transcription factor essential for skeletal muscle differentiation. A severe reduction in the number of fused myotubes is seen in myogenin-null mice, and the expression of genes characteristic of differentiated skeletal muscle is reduced. Additionally, sternebrae defects are seen in myogenin-null mice, a secondary defect in the sternal cartilage precursors. Very little is known about the quantitative requirement for myogenin in muscle differentiation and thoracic skeletal development in vivo. In this thesis I describe experiments utilizing a mouse line harboring a hypomorphic allele of myogenin, generated by gene targeting techniques in embryonic stem cells. The nature of the hypomorphism was due to lowered levels of myogenin from this allele. In embryos homozygous for the hypomorphic allele, normal sternum formation and extensive muscle differentiation was observed. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable after birth. These results suggest skeletal muscle differentiation is highly sensitive to the absolute amounts of myogenin, and reveal distinct threshold requirements for myogenin in skeletal muscle differentiation, sternum formation, and viability in vivo. The hypomorphic allele was utilized as a genetically sensitized background to identify other components of myogenin-mediated processes. Using a candidate gene approach I crossed null mutations in MEF2C and MRF4 into the hypomorphic background and examined whether these mutations affected muscle differentiation and skeleton formation in the myogenin hypomorph. Although MEF2C mutation did not affect any phenotypes seen in the hypomorphic background, MRF4 was observed to be an essential component of myogenin-mediated processes of thoracic skeletal development. Additionally, the hypomorphic allele was very sensitive to genetic effects, suggesting the existence of mappable genetic modifiers of the hypomorphic allele of myogenin. ^

Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody in HIV-1 infected adults

Context: Despite tremendous strides in HIV treatment over the past decade, resistance remains a major problem. A growing number of patients develop resistance and require new therapies to suppress viral replication. ^ Objective: To assess the safety of multiple administrations of the anti-CD4 receptor (anti-CD4) monoclonal antibody ibalizumab given as intravenous (IV) infusions, in three dosage regimens, in subjects infected with human immunodeficiency virus (HIV-1). ^ Design: Phase 1, multi-center, open-label, randomized clinical trial comparing the safety, pharmacokinetics and antiviral activity of three dosages of ibalizumab. ^ Setting: Six clinical trial sites in the United States. ^ Participants: A total of twenty-two HIV-positive patients on no anti-retroviral therapy or a stable failing regimen. ^ Intervention: Randomized to one of two treatment groups in Arms A and B followed by non-randomized enrollment in Arm C. Patients randomized to Arm A received 10 mg/kg of ibalizumab every 7 days, for a total of 10 doses; patients randomized to Arm B received a total of six doses of ibalizumab; a single loading dose of 10 mg/kg on Day 1 followed by five maintenance doses of 6 mg/kg every 14 days, starting at Week 1. Patients assigned to Arm C received 25 mg/kg of ibalizumab every 14 days for a total of 5 doses. All patients were followed for safety for an additional 7 to 8 weeks. ^ Main Outcome Measures: Clinical and laboratory assessments of safety and tolerability of multiple administrations of ibalizumab in HIV-infected patients. Secondary measures of efficacy include HIV-1 RNA (viral load) measurements. ^ Results: 21 patients were treatment-experienced and 1 was naïve to HIV therapy. Six patients were failing despite therapy and 15 were on no current HIV treatment. Mean baseline viral load (4.78 log 10; range 3.7-5.9) and CD4+ cell counts (332/μL; range 89-494) were similar across cohorts. Mean peak decreases in viral load from baseline of 0.99 log10(1.11 log10, and 0.96 log 10 occurred by Wk 2 in Cohorts A, B and C, respectively. Viral loads decreased by >1.0 log10 in 64%; 4 patients viral loads were suppressed to < 400 copies/mL. Viral loads returned towards baseline by Week 9 with reduced susceptibility to ibalizumab. CD4+ cell counts rose transiently and returned toward baseline. Maximum median elevations above BL in CD4+ cell counts for Cohorts A, B and C were +257, +198 and +103 cells/μL, respectively and occurred within 3 Wks in 16 of 22 subjects. The half-life of ibalizumab was 3-3.5 days and elimination was characteristic of capacity-limited kinetics. Administration of ibalizumab was well tolerated. Four serious adverse events were reported during the study. None of these events were related to study drug. Headache, nausea and cough were the most frequently reported treatment emergent adverse events and there were no laboratory abnormalities related to study drug. ^ Conclusions: Ibalizumab administered either weekly or bi-weekly was safe, well tolerated, and demonstrated antiviral activity. Further studies with ibalizumab in combination with standard antiretroviral treatments are warranted.^

THE USE OF MULTIVARIATE ANALYSIS OF COVARIANCE IN EVALUATING THE COMPARABILITY OF LABORATORY DETERMINATIONS IN COOPERATIVE STUDIES

The role of clinical chemistry has traditionally been to evaluate acutely ill or hospitalized patients. Traditional statistical methods have serious drawbacks in that they use univariate techniques. To demonstrate alternative methodology, a multivariate analysis of covariance model was developed and applied to the data from the Cooperative Study of Sickle Cell Disease.^ The purpose of developing the model for the laboratory data from the CSSCD was to evaluate the comparability of the results from the different clinics. Several variables were incorporated into the model in order to control for possible differences among the clinics that might confound any real laboratory differences.^ Differences for LDH, alkaline phosphatase and SGOT were identified which will necessitate adjustments by clinic whenever these data are used. In addition, aberrant clinic values for LDH, creatinine and BUN were also identified.^ The use of any statistical technique including multivariate analysis without thoughtful consideration may lead to spurious conclusions that may not be corrected for some time, if ever. However, the advantages of multivariate analysis far outweigh its potential problems. If its use increases as it should, the applicability to the analysis of laboratory data in prospective patient monitoring, quality control programs, and interpretation of data from cooperative studies could well have a major impact on the health and well being of a large number of individuals. ^

Evaluation of memory B cells among perinatally HIV infected children from Houston Texas

Background: HIV associated B cell exhaustion is a notable characteristic of HIV viremic adults. However, it is not known if such alterations are present in perinatal HIV infected children, whose viral dynamics differs from those seen in adults. In the present study we perform an analysis of B cells subsets and measure antigen-specific memory B cells (MBC) in a pediatric HIV infected cohort. ^ Methods: Peripheral mononuclear cells (PBMC) of perinatal HIV infected individuals are characterized into naïve (CD21hi/CD27−), classic (CD27+), tissue like (CD21lo/CD27 −) and activated MBC (CD27+CD21− ) by FACS. A memory ELISPOT assay is used to detect antibody secreting cells. We measure total IgG and antibodies specific for influenza, HBV, mumps, measles, rubella and VZV. Memory was expressed as spot forming cells (SPC) /million of PBMC. Wilcoxon rank-sum was used to compare unpaired groups and linear regression analysis was used to determine predictors of B cell dysfunction ^ Results: 41 HIV perinatal infected children are included (51.2% females and 65.9% Black). Age at study is median (range) 8.78 years (4.39-11.57). At the time of testing they have a CD4% of 30.9 (23.2-39.4), a viral load (VL) of 1.95 log10 copies/ml (1.68-3.29) and a cumulative VL of 3.4 log10 copy × days (2.7-4.0). Ninety two percent of the children are on cARV for > 6 months. Overall, HIV+ children compared with controls have a significant lower number of IgG and antigen specific SFC. In addition, they have a lower proportion of classical MBC 12.9 (8.09-19.85) vs 29.4 (18.7-39.05); 0.01, but a significant higher proportion of tissue like memory MBC 6.01 (2.79-12.7) vs 0.99 (0.87-1.38); 0.003, compared with controls. Patients are parsed on VL (<400 and ≥ 400 copies/ml) with the objective to evaluate the effect of VL on B cell status. Patients with a VL ≥ 400 copies/ml have a significantly lower IgG, HBV, measles, rubella and VZV SPC compared with those with a VL < 400 copies/ml. There are no significant differences in B cell subpopulations between the groups. A moderate negative correlation was observed between the time of cARV initiation and the frequency of IgG memory B cells, suggesting that early initiation of cARV appears to lead to a better functionality of the IgG memory B cells (P=0.05). A statistically significant positive correlation was observed between the total number of IgG memory cells and the number of antigen-specific memory B cells/SPCs. Suggesting that the progressive recovery of the IgG memory B cell pull goes along with a progressive increase in the number of antigen-specific SPCs. ^ Conclusion: A pediatric cohort in overall good status with respect to HIV infection and on ART has defects in B cell function and numbers (reduced total and antigen specific MBC and increased tissue like and reduced classical MBC).^