Raphael Wilson

A Special Report: Four-year Study of a Boy with Combined Immune Deficiency Maintained in Strict Reverse Isolation from Birth

Summary: A 4-year study of a boy with combined immune deficiency is presented, and the impact of this disease on various aspects of his growth and development is examined. There is no evidence of immune deficiency in either parent or in the genetic background on the maternal side. Three children of a brother of the mothers father may have had immune deficiencies but two have grown to be teenagers with no problems. Another died. At autopsy, however, lymph nodes appeared normal. The deceased older brother had severe combined immune deficiency (SCID). The autopsy findings showed Pneumocystis carinii pneumonia to be the direct cause of death and these findings contributed to the diagnosis of SCID. After a successful germ-free birth, the male infant (DV) was placed in the isolator. Laboratory tests were normal except that the x-rays showed no thymic shadow, his absolute lymphocyte count ranged from 399–440/mm, and the lymphocytes showed no proliferative response to phytohemagglutinin (PHA). Specific tests showed the antibody-producing immune system and the cell-mediated immune system to be severely defective. The patients lymphocytes elicited positive responses by lymphocytes from father, mother, and sister. Subsequent search in national and international tissue-typing laboratories has shown four HLA matches but none has been nonreactive in mixed lymphocyte culture (MLC). therefore, this patient has remained in isolation to the present; now he is 4 years old.Approximately 35 species of microorganisms, mostly transient contaminants, have been isolated, taking into account that the same organism may have been identified under different names in different laboratories. Those isolated frequently and in sufficiently high concentration to indicate colonization have been speciated as follows: anaerobes—Propionibacterium acnes, Lactobacillus catenaforme (disappeared spontaneously), Bacteroides oralis ss. elongatus, Clostridium (perenne, hastiforme, bifermentans), Bacteroides clostridiiformis ss. clostridiiformis; aerobes—Alcaligenes faecalis (eradicated by antibiotics), Staphylococcus epidermidis, Enterobacter agglomerans, Micrococcus sp. subgroup 1, Bacillus pulvifaciens (disappeared spontaneously); yeasts—Candida (tropicalis, parapsilosis). Seven are considered to be probable components of the current autoflora: P. acnes, C. bifermentans, B. clostridiiformis ss. clostridiiformis, S. epidermidis, Micrococcus sp. subgroup 1, E. agglomerans, C. parapsilosis. No viruses or protozoa have been isolated. At age 3 years, the mean quantitation of anaerobic cells was 7.9 × 107 viable cells/g feces; this falls short of the mean anaerobic load from normal children. The mean aerobic concentration was 1.2 × 108 viable cells/g feces, not unlike normal children. Qualitatively his flora has abnormally few species and lacks those most common in normal subjects. This child has had no evidence of infection and has always been in excellent health even though some organisms which could be pathogenic under some circumstances have been isolated.Phagocytic functions, adenosine deaminase (ADA) levels, and serum complement levels were normal except that C1q was 30% of normal. Thymosin assays showed adult control subject 1/4, 10-year control subject 1/128, this patient 0. To age 47 months serum immunoglobulin (Ig) M levels were generally low and IgG was not detected. No serum IgA was detected until, at 39 months, assays indicated IgA at the lowest range of sensitivity of the agar plates. Ultracentrifugal analysis of serum revealed no 19s material at 24 months but at 36 months both 7s and 19s materials were present. At 44 months these fractions were still present and an abnormal 4s component had disappeared. Radial immunodiffusion assay at 44 months indicated the presence of IgD and at that time an IgM component of normal electrophoretic mobility was detected for the first time. Before injection of keyhole limpet hemocyanin (KLH) at 1 month antibody liter was 0–0. Antibody titers and skin tests after injection were negative and remained so after further antigen injections. At 11 months, on the fifth rechallenge, the patient had an erythematous reaction of 5 mm diameter but no significant antibody responses. Two typhoid antigen injections elicited no antibody response. Using the isolated leukocyte technique, lymphocytes showed minimal or no blastogenesis in response to PHA, poke-weed mitogen (PWM), or in MLC. Using the whole blood technique, transient, low positive responses (stimulation index (SI) range 4.1–9.7) to PHA were observed but not consistently maintained. At 3 years, purified T cells showed a notable response (SI 17.4) to PHA, but this was not obtained in subsequent experiments. Transfer factor (TF) was given to this patient between 10 and 16 months of age. In skin tests to C. albicans, purified protein derivative (PPD) and streptokinase-streptodornase (SK-SD) administered a day after TF injection, small areas of redness appeared early and faded rapidly. Addition of TF to lymphocyte cultures obtained before administration of TF caused them to respond to C. albicans and PPD (SI 8 for each); after injection of TF and skin tests to these antigens, similar responsiveness could not be induced by addition of TF. After a second dose of TF, before skin test to SK-SD, the lymphocytes responded to SK-SD in vitro with TF (SI 13) and without TF (SI 8) added to the cultures; after SK-SD skin tests, responses were no longer elicited under the same circumstances. During the first 2 years membrane-bound immunoglobulin (SmIg), bearing lymphocytes ranged from 50–100% of the total lymphocytes whereas the percentage of lymphocytes with cells forming rosettes with sheep erythrocytes (E-RFC) markers was low, about 3–12%. Between 2.5 and 4 years striking changes occurred, representing a shift toward a normal distribution (20–40% SmIg and 19–60% E-RFC). In electron microscopic studies, new type lymphocytes which appeared at 15 months increased in number until at 4 years they represented 93% of the lymphocytes. In contrast to cells from normal donors, complement (C3) receptor-bearing cells of this patient did not express significant direct cytotoxicity; however, lymphotoxin (LT) levels 3–4 times those of E-RFC either from patient or normal donors were detected. Consistent with the positive responses, C3 receptor-stimulated cells also produced leukocyte migration inhibition factor (MIF) activity (40 * 16%) greater than background (15%) level. No correlation was evident between fluctuations in absolute numbers of C3 receptor-bearing cells in peripheral blood and the presence of LT and MIF responses. The expression of LT and MIF indicated that this patient was capable of nonspecific host responses. The findings suggest an impairment of the ability of this SCID patient to expand or sustain functional subpopulations essential to immunologic responsiveness.Hematologic surveillance revealed no dramatic differences from other children with SCID. Absence of complicating infections indicated that the changes were probably attributable to the natural disorder. Thrombocytopenia appears to be related to the basic disease because occurrence and recovery did not relate in a consistent pattern with antibiotic therapy, other therapy, or bacterial contamination. Peri~heral blood counts followed a pattern consistent with previously described cases. At 3 months of age the patient showed scalp changes, loss of hair, and rough skin. He was placed on an iron-fortified formula and vitamins. The scalp problems soon cleared up. Another episode of hair loss and dry skin occurred at 15 months. He had not been given his vitamin supplements. Vitamins were given again. Subsequently scalp problems improved. This is a child who has been on low cholesterol intake from birth, yet there is no malfunction of the nervous system or delay in myelinization. At age 2 years he was excreting a larger percentage of primary bile acids (70%) than control subjects (30%). This indicated a reduced activity of bacterial flora. DV excreted 99.4% of his neutral lipids as cholesterol with a trace of coprostanol whereas control subjects of 1.5 years or less showed 95% cholesterdl~with about 5% coprostanol.When he was 5 months, 24 days of age, using the BayleyScales of Infant Development, the Mental Index was 116 (6.5- 7.5 months) and the Psychomotor Index was 112 (6.5-7.5 months). When he was 3 years old, potential intellectual endowment was estimated to be 1-2 years above his chronologic age. He showed unusual ability in the discrimination and recognition of geometrical shapes of objects in his environment. Evaluation of general ego functioning showed rejection of some popular responses, a healthy awareness of color nuances, a rather low prdductivity relative to his intellectual potential, but no apparent idiosyncratic or dereistic quality in his ideation or concept formation. At age 12 months, the child showed a marked deficit in receptive and expressive language skills. Implementation of a program of language stimulation resulted in rapid and consistent improvement. Recent objective testing revealed above average speech and language abilities, except for a mild, persistent deficit in receptive and expressive vocabulary which was related to his atypicai environment.Psychiatric evaluation in this patient demonstrates that it is possible to rear a child, under conditions of strict reverse isola tion, who can respond with normal affective, cognitive, and intellectual ability to age 4 years.Speculation: The continued maintenance of this patient in a gnotobiotic state has provided opportunity for serial studies in an uncomplicated disease state.Although he has not remained germ free, this technology has been successful in preventing infection for 4 years in a child who, otherwise, would have been overwhelmed with infection. In addition, the studies have shown that the significantly simpler microflora in the early part

Evidence for a toxic substance of bacterial origin in the blood of irradiated mice.

Germfree mice have been used to investigate the presence, nature, and origin of toxins suspected of circulating in the blood following high doses of wholebody radiation. The serum bound iron (SBI) ...

Increased Intestinal Mucosal Turnover and Radiosensitivity to Supralethal Whole-Body Irradiation Resulting from Cholic Acid-Induced Alterations of the Intestinal Microecology of Germfree CFW Mice'

The prolonged mean survival time of germfree mice, compared to conventional mice, after exposure to 1000-10,000 rad whole-body irradiation has been postulated to be a function of an increased turno...

Antibiotic radioprotection of mice exposed to supralethal whole-body irradiation independent of antibacterial activity.

Oral administration of streptomycin, kanamycin, neomycin, or gentamicin to specific pathogen-free C57 x Af mice in their drinking water (4 mg/ml) for 2 weeks before supralethal whole-body irradiation very significantly prolonged their mean survival times (8.2 to 8.9 days vs 6.9 for controls) to values which exceed those reported for germ-free mice (7.3 days). The total fecal concentrations of aerobes and anaerobes were reduced by kanamycin, neomycin, and gentamicin. Streptomycin reduced the anaerobes significantly, but not the aerobes. Unlike germ-free mice, these antibiotic-treated mice did excrete free bile acids, products of bacterial action. Oral antibiotic treatment was ineffective in altering the transit time of the intestinal mucosal cells. Previously reported studies had indicated a correlation between decreased transit time and increased survival after irradiation. No significant correlation between mean survival time after irradiation and mucosal transit time was observed. The data demonstrate that certain antibiotics alter the character of the intestinal bacterial flora and increase protection against supralethal doses of whole-body irradiation. It is concluded that the mechanisms of radioresistance in antibiotic-treated mice and germ-free mice are different and that in both groups radioresistance is the result of more than elimination of postirradiation infection.

Homograft Rejection by Neonatally Thymectomized Germ-free Mice

Summary A response to tissue transplantation was elicited in germ-free neonatally thymectomized CFW and C3H mice by challenge with homotransplantations of methylcholanthrene-induced tumor cells and neoplastic lymph node cells. Germ-free thymectomized mice of both CFW and C3H strains accepted all isologous transplants and rejected all homologous transplants with only 1 exception. When these thymectomized mice were irradiated, their immune response seemed to be effectively depressed and homologous transplants made 3 days after irradiation were accepted by both strains. In CFW thymectomized and irradiated mice the tumor enlarged and eventually killed the mouse, but in C3H thymectomized and irradiated mice the tumor did not enlarge, but persisted until 6 weeks, when the experiment was terminated and the animals were sacrificed and autopsied. However, when transplantation of C3H tumors to CFW mice was delayed until 2 months after irradiation to permit recovery of immunologically active tissues, most of the tumors were rejected after a brief period of acceptance. Results of transplantation of neoplastic lymph node cells from C3H mice infected with leukemia were comparable to those from the tumor transplants. Both germ-free and germ-free thymectomized CFW mice were able to reject homologous lymph node cells from C3H mice; all but 1 of the C3H control mice developed leukemia. Neonatal thymectomy alone does not deprive CFW and C3H mice of the ability to reject tumor homotransplants, nor does exposure to 300 R of X-radiation abolish this response in these thymectomized mice. The assault by microorganisms on the conventional mouse at a time when it has been deprived of most of its lymphoid tissue appears to overtax its immunologic defense and to induce a debilitated state in which it cannot successfully respond to a homotransplant.

A Novel Algorithm for Simplification of Complex Gene Classifiers in Cancer

The clinical application of complex molecular classifiers as diagnostic or prognostic tools has been limited by the time and cost needed to apply them to patients. Using an existing 50-gene expression signature known to separate two molecular subtypes of the pediatric cancer rhabdomyosarcoma, we show that an exhaustive iterative search algorithm can distill this complex classifier down to two or three features with equal discrimination. We validated the two-gene signatures using three separate and distinct datasets, including one that uses degraded RNA extracted from formalin-fixed, paraffin-embedded material. Finally, to show the generalizability of our algorithm, we applied it to a lung cancer dataset to find minimal gene signatures that can distinguish survival. Our approach can easily be generalized and coupled to existing technical platforms to facilitate the discovery of simplified signatures that are ready for routine clinical use.

Negative regulation of initial steps in skeletal myogenesis by mTOR and other kinases

The transition from a committed progenitor cell to one that is actively differentiating represents a process that is fundamentally important in skeletal myogenesis. Although the expression and functional activation of myogenic regulatory transcription factors (MRFs) are well known to govern lineage commitment and differentiation, exactly how the first steps in differentiation are suppressed in a proliferating myoblast is much less clear. We used cultured mammalian myoblasts and an RNA interference library targeting 571 kinases to identify those that may repress muscle differentiation in proliferating myoblasts in the presence or absence of a sensitizing agent directed toward CDK4/6, a kinase previously established to impede muscle gene expression. We identified 55 kinases whose knockdown promoted myoblast differentiation, either independently or in conjunction with the sensitizer. A number of the hit kinases could be connected to known MRFs, directly or through one interaction node. Focusing on one hit, Mtor, we validated its role to impede differentiation in proliferating myoblasts and carried out mechanistic studies to show that it acts, in part, by a rapamycin-sensitive complex that involves Raptor. Our findings inform our understanding of kinases that can block the transition from lineage commitment to a differentiating state in myoblasts and offer a useful resource for others studying myogenic differentiation.

Potential pitfalls of mass spectrometry to uncover mutations in childhood soft tissue sarcoma: A report from the Children’s Oncology Group

Mass spectrometry-based methods have been widely applied – often as the sole method – to detect mutations in human cancer specimens. We applied this approach to 52 childhood soft tissue sarcoma specimens in an attempt to identify potentially actionable mutations. This analysis revealed that 25% of the specimens harbored high-confidence calls for mutated alleles, including a mutation encoding FLT3I836M that was called in four cases. Given the surprisingly high frequency and unusual nature of some of the mutant alleles, we carried out ultra-deep next generation sequencing to confirm them. We confirmed only three mutations, which encoded NRASA18T, JAK3V722I and METR970C in three specimens. Beyond highlighting those mutations, our findings demonstrate potential pitfalls of primarily utilizing a mass spectrometry-based approach to broadly screen for DNA sequence variants in archived, clinical-grade tumor specimens. Duplicate mass spectrometric analyses and confirmatory next generation sequencing can help diminish false positive calls, but this does not ameliorate potential false negatives due in part to evaluating a limited panel of sequence variants.

Abstract PR02: Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is composed of skeletal myoblast-like cells that have lost the capacity to terminally differentiate. This suggests that RMS cells may contain a factor that blocks normal muscle differentiation. Because cell cycle arrest is coupled to muscle differentiation, identifying putative negative regulators of differentiation could lead to novel therapeutic approaches aimed at fostering terminal differentiation. To gain insight into the events that normally trigger the initial phase of muscle differentiation, we carried out a high content cell-based screen using detection of Myogenin, an early marker of muscle differentiation, as a readout that we measured by automated fluorescence microscopy. We focused on identifying kinases that hinder this developmental transition because of the potential to develop pharmacological inhibitors of “hit” kinases. Using an siRNA library targeting over 600 kinases, we identified 56 as putative negative regulators of myogenic differentiation: 43 with and 19 without PD332991, a Cdk4/6 inhibitor included as a sensitizer. Network analysis showed that 47% of the hits identified without the sensitizer, including SRC family kinases Src and Fyn , were just one interaction node away from MyoD- or Mef2-family myogenic regulatory factors. Although some of the hit kinases were previously implicated in myogenesis, many others were not. Further, 9 (16%) of the hits are causally linked with cancer, based on KEGG and COSMIC databases. Pathway analyses highlighted certain cancer-associated pathways, like EPHA/Ephrin B signaling, the MAPK kinase pathway, de novo pyrimidine synthesis, and mTOR signaling. Among the hits relevant to RMS, Mtor was particularly interesting because this gene encodes a kinase regarded to positively regulate skeletal muscle differentiation. We confirmed our screen findings that Mtor blocks muscle differentiation by showing that its knockdown increases the transcription of a panel of muscle-specific genes in an established myoblast cell line and primary mouse myoblasts. Induction of muscle genes by Mtor knockdown correlated with G 0 /G 1 cell cycle arrest that normally accompanies differentiation. Rapamycin mimicked the effects of Mtor knockdown on muscle gene expression and cell proliferation, implying a role for mTorc1. Finally, preliminary analysis of RMS gene expression data demonstrated that higher expression of muscle differentiation genes correlates with improved survival. These data highlight the potential for mTOR inhibitors to foster the expression of muscle specific genes, pushing the myoblast-like tumor cells to a more differentiated state. Altering their biology in this way may improve outcome. On-going efforts are exploring the mechanisms acting downstream of Mtor to impede muscle differentiation and directly evaluating pro-differentiation effects of mTOR inhibition in RMS models. This abstract is also presented as Poster A1. Citation Format: Raphael A. Wilson, Jing Liu, Lin Xu, Yanbin Zheng, Stephen X. Skapek. Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR02.