Atm heterozygosity cooperates with loss of Brca1 to increase the severity of mammary gland cancer and reduce ductal branching.

The role of homozygous ataxia telangiectasia mutated (ATM) mutations in familial and sporadic forms of cancer is well established, but the contribution of ATM heterozygosity to mammary gland and other cancers has been controversial. To test the effect of Atm heterozygosity on mammary gland cancer, mice with complete loss of exon 11 of Brca1 specifically in mammary epithelium (Brca1-MG-Deltaex11) were studied in either Atm heterozygous or Atm wild-type backgrounds. Targeted deletion of Brca1 in mammary epithelium resulted in carcinomas and adenocarcinomas of varying histology with long (>9 months) latency. Latency to tumorigenesis was found to be unchanged in the Brca1-MG-Deltaex11;Atm heterozygous mice compared with Brca1-MG-Deltaex11;Atm wild-type mice. However, the mice displayed variable tumor severity and differences in mammary tissue development. Mammary tumors from Brca1-MG-Deltaex11;Atm heterozygous mice were anaplastic and undifferentiated in all 20 tumors tested, whereas tumors from mice that were Brca1-MG-Deltaex11 but wild-type for Atm displayed variable histologic profiles, with some anaplastic tumors and other differentiated and less invasive tumor types. Previously reported developmental defects for Brca1-deficient mice were also observed in our model with and without Atm heterozygosity, but Brca1-MG-Deltaex11;Atm heterozygous mice displayed decreased ductal branching during puberty, a phenotype that was not observed in Brca1-MG-Deltaex11;Atm wild-type mice. Our results provide evidence that Atm heterozygosity influences severity of mammary gland tumors in the Brca1-MG-Deltaex11 tumor-prone mouse and suggest that this mutation leads to a newly characterized developmental defect during glandular maturation.

Aberrant recombination involving the granzyme locus occurs in Atm-/- T-cell lymphomas.

Ataxia telangiectasia (A-T) is an autosomal recessive disease caused by loss of function of the serine/threonine protein kinase ATM (ataxia telangiectasia mutated). A-T patients have a 250-700-fold increased risk of developing lymphomas and leukemias which are typically highly invasive and proliferative. In addition, a subset of adult acute lymphoblastic leukemias and aggressive B-cell chronic lymphocytic leukemias that occur in the general population show loss of heterozygosity for ATM. To define the specific role of ATM in lymphomagenesis, we studied T-cell lymphomas isolated from mice with mutations in ATM and/or p53 using cytogenetic analysis and mRNA transcriptional profiling. The analyses identified genes misregulated as a consequence of the amplifications, deletions and translocation events arising as a result of ATM loss. A specific recurrent disruption of the granzyme gene family locus was identified resulting in an aberrant granzyme B/C fusion product. The combined application of cytogenetic and gene expression approaches identified specific loci and genes that define the pathway of initiation and progression of lymphoreticular malignancies in the absence of ATM.

Unraveling human cancer in the mouse: recent refinements to modeling and analysis.

The ability to manipulate the mouse genome has made the mouse the primary mammalian genetic model organism. It has been possible to model human cancer in the mouse by overexpressing oncogenes or inactivating tumor suppressor genes, and these experiments have provided much of our in vivo understanding of cancer. However, these transgenic approaches do not always completely and accurately model human carcinogenesis. Recent developments in transgenic and knockout approaches have improved the accuracy of modeling somatic cancer in the mouse and analyzing the genomic instability that occurs in murine tumors. It is possible to use retroviral gene delivery, chromosome engineering and inducible transgenes to selectively manipulate the genome in a more precise spatial and temporal pattern. In addition, the development of powerful cytogenetic tools such as spectral karyotyping, fluorescence in situ hybridization and comparative genome hybridization have improved our ability to detect chromosomal rearrangements. Finally, global patterns of gene expression can be determined by microarray analysis to decipher complex gene patterns which occur in cancers. Several of these advances in mouse modeling of human cancer are discussed in this review.

Production of factors with B-cell growth and differentiation activities by peripheral blood mononuclear cells from patients with hypogammaglobulinemia.

BACKGROUND: The maturation of normal B lymphocytes proceeds through a growth phase and a differentiation phase. These two phases appear to be under the influence of mediators released by immune cells, B-cell growth factor(s), which induce proliferation of B cells; and B-cell differentiation factor(s), which induce B-cell differentiation. METHODS: We analyzed the ability of peripheral blood mononuclear cells from patients with hypogammaglobulinemia to produce B-cell growth factor and B-cell differentiation factor activity in comparison with normal peripheral blood mononuclear cells. RESULTS: Of 27 patients tested, 26 had normal production of B-cell growth factor activity. A quantitative but not absolute defect in B-cell growth factor production was demonstrable in one boy with hypogammaglobulinemia. Interleukin-2 and interleukin-4 levels, as determined antigenically in these supernatants, had a similar distribution pattern from patients' or from control peripheral blood mononuclear cells; that is, undetectable levels of interleukin-2 were produced by cells from 4 of 16 patients tested and from 4 of 13 control subjects, and undetectable levels of interleukin-4 produced by cells from 6 of 16 patients and 4 of 13 control subjects. B-cell differentiation factor activity was absent in only one child tested but present in all other patients. Two patients had quantitatively low secretion of B-cell differentiation factor, but all others were within normal range. The two patients with quantitatively depressed B-cell differentiation factor activity had normal levels of B-cell growth factor activity, interleukin-2, and interleukin-4 produced from their cells. CONCLUSION: Peripheral blood mononuclear cells from the majority of patients with hypogammaglobulinemia appear to have the capacity to produce B-cell growth factors and B-cell differentiation factor activity in vitro.

Assessment of cisplatin-induced ototoxicity using derived-band ABRs.

Ototoxicity is an adverse side effect of numerous therapeutic agents (amino-glycoside antibiotics, blood chelating agents, diuretics and oncologic drugs) used in treatment of both adult and pediatric patients. Recently, there has been increasing interest in using the auditory brainstem response (ABR) to detect both short-term effects of ototoxicity in adults and long-term effects of drug administration on neonates and children. Since click ABRs have relatively poor frequency selectivity they best approximate the pure-tone hearing threshold in the 2000-4000 Hz frequency range. Hearing loss above or below that frequency range can be present without producing significant abnormalities in the ABR waveform parameters. Frequency-specific ABRs can be obtained using the derived response technique. The purpose of this study was to investigate early cisplatin ototoxicity using both the broadband click and derived ABR and to monitor progressive hearing loss with repeated drug trials in 18 patients studied over a 2-year period. ABRs were obtained serially prior to and following intravenous administration of cisplatin. Derived ABRs were found to be more sensitive than broadband click ABR in detecting early high-frequency hearing loss. For click ABRs, the cumulative dosage of cisplatin at age of ABR examination was correlated with hearing loss in only those patients under 3 years of age. No significant correlation was found between cumulative cisplatin dosage when tested and degree of hearing loss in those patients over 3 years of age.

Genetic markers and inflammatory bowel disease: immunoglobulin allotypes (GM, KM) and protease inhibitor.

We have studied immunoglobulin allotypes (GM and KM) in 101 patients with Crohn's disease, 51 patients with ulcerative colitis, and 99 healthy local blood donor controls. In addition, protease inhibitor (PI) types were examined in a random subset of patients and in all controls. No significant differences were found between Crohn's disease patients and controls, or between ulcerative colitis patients and controls, in the frequencies of GM phenotypes, GM haplotypes, KM phenotypes, or PI phenotypes.

Passive and active immunization against hepatitis B virus infection: optimal scheduling in healthy young adults.

Recent in vitro data suggest that an enhanced humoral response to hepatitis B surface antigen (HBsAg) may be achieved when HBsAg is administered to monocyte/lymphocyte mixtures in the presence of antibody to HBsAg (Anti-HBs). In order to determine whether the same response can be achieved in vivo and whether this phenomenon can be used to augment an individual's response to hepatitis B virus (HBV) vaccination, serum antibody levels to hepatitis B surface antigen (anti-HBs) were determined in five groups of healthy young volunteers receiving passive hepatitis B immunoglobulin (HBIG) at various time intervals around a fixed HBV vaccination schedule. Thus HBIG (0.06 ml/kg) was administered to individuals in group I at -4 weeks; group II: -2 weeks; group III: simultaneously with vaccination; group IV; +2 weeks; and group V: +4 weeks following the onset of active immunization. A sixth group of volunteers received HBIG alone. The results of the study indicated that following the initial vaccination, individuals receiving HBIG prior to vaccination responded more often and with slightly higher anti-HBs levels than those receiving HBIG simultaneously or following the onset of vaccination. These differences, however, became less prominent and ultimately non-existent with subsequent boosts of the vaccine. The study also demonstrated that males and individuals over thirty do not respond to HBV vaccination as well as females or those under thirty.

Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.

Efficacy of commercial condoms in the prevention of hepatitis B virus infection.

Tips of synthetic and natural condoms were filled with serum samples containing either hepatitis B virus, herpes simplex virus, or cytomegalovirus, then fit over an 8-in. mechanical vibrator and inserted vibrating into sterile bath solutions for 30 min. Phosphorus 32-labeled hepatitis B and cytomegalovirus molecular probes and viral culture techniques for herpes simplex and cytomegalovirus were used to determine whether leakage of virus had occurred into the surrounding bath solutions. Natural condoms allowed leakage of hepatitis B virus but not herpes simplex virus or cytomegalovirus, whereas synthetic condoms prevented leakage of all viruses. These results suggest that natural condoms might not be effective in preventing sexually transmitted hepatitis B virus infection.

The nature of antibody to hepatitis B surface antigen in high-risk persons negative for other hepatitis B viral markers.

Persons with antibody to hepatitis B surface antigen (anti-HBs) activity in their serum that is entirely immunoglobulin M (IgM) in nature appear not to be protected against hepatitis B virus infection. Because commercially available anti-HBs kits do not distinguish between IgM and immunoglobulin G anti-HBs activity, the question of whether or not to immunize high-risk persons with anti-HBs alone has recently been the subject of much controversy. The present study in Calgary and Winnipeg, 1984-1985, documents the nature of anti-HBs activity in sera from three groups of "high-risk" persons. Group I consisted of 26 health care employees positive for anti-HBs but negative for other hepatitis B serologic markers. Group II consisted of 26 members of high-risk ethnic populations who were also positive for anti-HBs alone. Group III consisted of 29 persons from both populations (16 health care workers and 13 members of high-risk ethnic populations) positive for both anti-HBs and antibody to hepatitis B core antigen (anti-HBc). The majority of persons in groups I and II (58% and 50%, respectively) had low levels of anti-HBs activity (sample/negative control ratio (ratio unit) less than 10), as compared with only three of group III (p less than 0.00001 and 0.0001, respectively). Of the remaining persons with presumably protective levels of anti-HBs (ratio unit greater than or equal to 10), treatment with 0.2 mM dithiothreitol, an agent that completely destroys IgM immunoglobulin activity, resulted in less inhibition of anti-HBs activity in sera from groups I and II (48.2 +/- 31.1 and 51.9 +/- 19.5, mean +/- standard deviation, respectively) than group III sera (69.9 +/- 22.0) (p less than 0.05). In no case was anti-HBs activity completely eliminated following dithiothreitol treatment. The results of this study indicate that anti-HBs activity in the majority of high-risk persons with presumably protective levels of anti-HBs is not exclusively IgM in nature.

Humoral immune response in patients with hemophilia.

Hemophiliacs require frequent infusions of allogeneic proteins to control bleeding. Previous reports have demonstrated that thymus-derived lymphocytes (T cells) from hemophiliacs are antigenically primed to the lyophilized antihemophilic factor and that natural killer cells from hemophiliacs demonstrate impaired response to interferon-beta and -gamma Some aspects of the humoral immune response were investigated in eight patients who require large amounts of Factor VIII. Polyclonal hypergammaglobulinemia was detected in six patients and seven had elevated titers of autoantibodies of various specificities. There was no evidence of impaired concanavalin A-inducible T-suppressor cell activity. Polyclonal immunoglobulin secretion secondary to pokeweed mitogen in vitro was elevated in three of eight patients and depressed in five. Spontaneous production of both B-cell growth and differentiation factors (BCGF and BCDF) was elevated but mitogen-induced production was impaired. These data demonstrate that the humoral immune response of hemophiliacs may be chronically stimulated, thus impairing their ability to respond to new antigens such as viruses.

Fatal Aspergillus pneumonia in chronic granulomatous disease.

A 20-year-old man died from Aspergillus pneumonia three weeks after heavy exposure to grain dust. Lung biopsy and autopsy demonstrated a distinctive form of suppurating granulomatous bronchopneumonia caused by Aspergillus fumigatus, which was the sole agent cultured from the tissue. The liver and lymph nodes contained pigmented lipid histiocytes characteristic of chronic granulomatous disease, and subsequently both of the patient's brothers were found to have X-linked chronic granulomatous disease. The authors suggest that this morphologic expression of Aspergillus pneumonia should raise a suspicion of neutrophil dysfunction or chronic granulomatous disease.

Familial concurrence of pauciarticular syndrome and systemic sclerosis.

We observed a sibship of two children who had the syndrome of early-onset pauciarticular arthritis with anti-nuclear antibodies; their paternal grandmother died of progressive systemic sclerosis. Since the early-onset pauciarticular syndrome is very rare or non-existent in adults, the findings in this kindred pair raise the possibility that this disorder and adult systemic sclerosis may share some genetic or other etiological factors.

T lymphocytes from hemophiliacs proliferate after exposure to factor VIII product.

Chronic exposure of hemophiliacs to allogeneic proteins via factor VIII concentrate for control of bleeding could lead to a state of chronic antigen stimulation. Immune function from eight hemophiliacs requiring large amounts of factor VIII concentrate was assessed. It was found that 5 of 8 had a positive blastogenic response to low concentrations of factor VIII in a 7-day thymidine uptake assay. Separation of lymphocyte subpopulations indicated that the reactivity was contained in the T cell-enriched fraction. There was no blast transformation noted secondary to the factor VIII product in 20 controls tested in a 7-day assay nor was there any mitogenic effect of the factor VIII in a 3-day assay. In fact, high concentrations of factor VIII impaired that T lymphocytes from hemophiliacs are antigenically primed to some constituent in the lyophilized factor VIII concentrate.

Natural killer cell activity from hemophiliacs exhibits differential responses to various forms of interferon.

Patients with hemophilia are at risk for the development of acquired immunodeficiency syndrome (AIDS). Patients with AIDS have recurrent infections and/or malignancy and altered immune response, including decreased T lymphocyte counts, decreased T helper lymphocytes, defective T cell blastogenesis, hypergammaglobulinemia, defective natural killer (NK) activity and impaired response of NK to interferon-beta (IFN-beta). It is feasible that chronic antigen stimulation with subsequent release of interferon could be related to the impaired NK reactivity to IFN-beta of patients with AIDS. Because hemophiliacs are subjected to chronic antigen stimulation secondary to the administration of foreign protein, the reactivity of NK cells from patients with hemophilia to IFN-alpha, IFN-beta and IFN-gamma was studied. Eight patients with hemophilia requiring high levels of clotting factor replacement were assessed. Three patients were antibody positive to HTLV-III. All had normal baseline NK cell function. In the first set of experiments, all patients responded normally to in vitro IFN-alpha by increasing NK activity, but four patients had significant failure and two had mild impairment in NK response to IFN-beta. This latter observation was particularly evident at very low concentrations of IFN. In repeated experiments, seven of eight had impaired NK response to IFN-beta and IFN-gamma but normal response to IFN-alpha. Only one patient's NK cells responded better to IFN-gamma. There was no obvious correlation of these findings to antibody status to HTLV-III. Chronic antigen stimulation and the modulation of interferon receptors are discussed as possible mechanisms that could produce these findings.

Antinuclear, anticytoplasmic, and anti-Sjogren's syndrome antigen A (SS-A/Ro) antibodies in female blood donors.

A study of 2500 sera from female blood donors between the ages of 20 and 50 years was undertaken to determine the frequency of antinuclear (ANA), anticytoplasmic (ACA), and antimitochondrial (AMA) antibodies. When sera were tested by immunofluorescence (IF) on HEp-2 cells, 15.9 and 1.1% had ANA titers greater than 1/20 and 1/80, respectively. Analysis of these sera for autoantibody specificity showed: 1.5% antinucleolar, 1.0% anti-nuclear matrix, 0.2% anti-mitotic spindle apparatus, and 0.2% anti-primary biliary cirrhosis nuclear antigen. AMA titers of greater than 1/80 were seen in 2.5% and AMA titers greater than 1/160 were seen in 1.0%. None of the sera had anti-double stranded DNA. Testing of an additional 2500 sera for anti-Sjogren's Syndrome antigen A (anti-SS-A/Ro) revealed a frequency of 22/5000 (0.44%) with the highest frequency (0.72%) being in the 45-50 age group and a relatively high frequency (0.58%) in the 20-24 age group.

Transient immunodeficiency during asymptomatic Epstein-Barr virus infection.

In vivo and in vitro humoral and cellular immune responses were studied in a 2 1/2-year-old girl immediately before, during, and after an asymptomatic infection with Epstein-Barr virus. During the acute EBV infection, the patient's peripheral blood mononuclear cells were deficient in immunoglobulin synthesis and suppressed the in vitro immunoglobulin synthesis of normal allogeneic cells. In vitro mitogen transformation of lymphocytes was reduced. In vivo antibody responses to the T cell-dependent antigens bacteriophage phi X 174 and Keyhole limpet hemocyanin were markedly depressed. These studies suggest that suppressor cells induced during acute EBV infection not only suppress immunoglobulin synthesis in vitro, but also interfere with in vivo antibody synthesis.

Severe recurrent bacterial infections associated with defective adherence and chemotaxis in two patients with neutrophils deficient in a cell-associated glycoprotein.

We studied two patients with delayed umbilical cord detachment, recurrent bacterial infections, inability to form pus, rapidly progressive periodontitis, and persistent leukocytosis. The phagocytes of both patients were strikingly abnormal in their ability to adhere to surfaces. The adherence of polymorphonuclear leukocytes to endotoxin-coated glass coverslips, glass beads, or nylon wool was markedly reduced. Scanning electron microscopy of the few adherent polymorphonuclear leukocytes from both patients showed a failure to flatten and form fine pseudopods. In vivo polymorphonuclear leukocyte and monocyte chemotaxis assessed by skin window and skin chamber methods was dramatically impaired, and in vitro chemotaxis was severely depressed. Chemiluminescence of zymosan- but not phorbol-stimulated polymorphonuclear leukocytes was markedly reduced. Allogeneic polymorphonuclear leukocytes transfused into these patients functional normally, indicating that the defect is intrinsic to the cells and not a secondary phenomenon. A 180-kilodalton glycoprotein normally present in the particulate fraction of polymorphonuclear leukocytes was found to be completely absent in Patient 1 and present in low concentration in Patient 2. We postulate that the glycoprotein deficiency interferes with the migration of polymorphonuclear leukocytes from the bloodstream into the interstitial space and to the site of infection.