Bette A. Pancake

Rag mutations reveal robust alternative end joining

Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1–Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, ‘alternative NHEJ’, appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell’s overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.

HTLV tax and Mycosis Fungoides

To the Editor: Although a causal relation between human T-cell lymphotropic retrovirus type I and type II (HTLV-I and HTLV-II) and adult T-cell leukemia has been well established, the association b...

The difficulty of detecting HTLV-I proviral sequences in patients with mycosis fungoides

Although most patients with cutaneous T cell lymphomas, including mycosis fungoides (MF) and its leukemic variant, the Sézary syndrome, are seronegative for antibodies to the human T cell lymphotropic viruses (HTLV-I/II), it has recently been shown that > 95% of such patients harbor proviral DNA sequences related to the region of the HTLV genome that encodes the transregulatory/transforming gene, tax. However, the demonstration of HTLV sequences, even after amplification by polymerase chain reaction (PCR), has not been universally successful, and some investigators continue to question this observation. In an effort to resolve this controversy, we have compared published methodologies that have been less successful with techniques currently used in this laboratory. Major differences were found in (a) the nature of the cells used [freshly isolated versus cultured peripheral blood mononuclear cells (PBMC)] and (b) the methods used to prepare samples for PCR (whole cell lysates versus DNA extracts). PBMC from 10 different MF patients and the healthy daughter of 1 of the patients were subjected to comparative analyses. While all of the PBMC lysates were positive, the DNA extract from only one of these individuals revealed HTLV tax sequences. Studies were also conducted comparing cell lysates and DNA extracts of cultured cells derived from tax sequence-positive PBMC from seven different MF patients. The cells from four of the seven were shown to have retained tax sequences after varying times in culture, when whole-cell lysates were used as targets for PCR amplification and Southern analysis, whereas none of the DNA extracts were positive. It appears that the use of whole-cell lysates instead of DNA extracts and the use of fresh instead of cultured cells greatly enhance the ability to detect HTLV-1 tax sequences in specimens from MF patients.

Hypopigmented Mycosis Fungoides Associated With Human T Cell Lymphotropic Virus Type I Tax in a Pediatric Patient

* Abbreviations: MF = : mycosis fungoides • HTLV-I = : human T cell lymphotropic virus type I • NYUMC = : New York University Medical Center • UVB = : ultraviolet B • PBMC = : peripheral blood mononuclear cells • PCR = : polymerase chain reaction The cutaneous T cell lymphoma, mycosis fungoides (MF), is characterized by proliferation of atypical lymphocytes that preferentially home to the skin. Usually, afflicted patients are middle-aged and present with erythematous patches and plaques. Although often indolent, the disease may progress to a tumor stage and involve viscera. The present report concerns a 7-year-old black boy, who was born in Grenada and who presented with a 3-year history of slowly developing, hypopigmented patches scattered over his buttocks, legs, and arms involving approximately 20% of his body surface. He was serologically negative for antibodies to human T cell lymphotropic virus types I/II (HTLV-I/II). The young age of this patient, the hypopigmentation of his lesions, his origin from a region of the world where HTLV-I is endemic, and our previous studies showing an association of HTLV-I tax with MF,1 ,2 prompted us to investigate whether the patient and/or his first-degree relatives were infected with this virus. While the patients healthy mother proved to be a carrier of HTLV-I, with antibodies to the virus when tested by routine serologic methods, the patient, like other patients with MF, as well as his healthy younger brother, harbored only deleted HTLV-I proviral sequences. The possible implications of this observation are discussed. A 7-year-old black boy, born in Grenada, was referred to the Dermatology Service at New York University Medical Center (NYUMC) because of a 3-year history of hypopigmented macules and patches covering about 20% of his body (Fig 1). The hypopigmented lesions were not pruritic, scaly, or palpable to touch. He was otherwise well. His past medical history was relevant only in that he was born by spontaneous vaginal delivery and that he had been breastfed. Physical examination revealed no lymphadenopathy or organomegaly. Routine blood counts and biochemistries were all within normal range. He was serologically …

Localization of HTLV-I tax proviral DNA in mononuclear cells

The tax sequence of HTLV-I is demonstrable in the skin and blood mononuclear cells of patients with mycosis fungoides, as well as in the mononuclear leukocytes of some healthy blood donors, but was not demonstrable when PCR/Southern analyses were carried out on preparations of high-molecular-weight genomic DNA. Therefore, it was postulated that tax DNA may not be integrated. To investigate this possibility fluorescence in situ hybridization was carried out on cells arrested in metaphase, using a probe containing the HTLV-I tax proviral DNA full-length open reading frame coding sequence. While metaphases prepared from C91PL cells, a cell line infected with HTLV-I, showed an abundance of chromosome-associated as well as extra-chromosomal signals, metaphases prepared with blood mononuclear cells from healthy tax sequence positive donors did not reveal any tax DNA associated with chromosomes. Such signals were readily detected extra-chromosomally. Although it has been demonstrated that transactivation of genes by gene products encoded by extra-chromosomal DNA may have nosocomial implications, whether transactivation by p40 tax generated from extra-chromosomal tax sequences is responsible for the development of neoplasia remains to be investigated.

Transmission of Human T-Cell Lymphotropic Virus Type 1 Tax to Rabbits by tax-Only-Positive Human Cells

ABSTRACT The human T-cell lymphrotropic virus type 1 (HTLV-1) is causally related to adult T-cell leukemia and lymphoma and the neurodegenerative diseases tropical spastic paraparesis and HTLV-1-associated myelopathy. In the United States the prevalence of infection has been estimated to range from 0.016 to 0.1% on the basis of serologic tests for antibodies to the viral structural proteins. Blood from donors positive for antibodies to HTLV-1 or HTLV-2 is not used for transfusion. However, patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) are HTLV-1 and -2 seronegative yet harbor proviral sequences identical to those that encode the HTLV-1 transactivating and transforming gene product p40tax in their peripheral blood mononuclear cells (PBMCs), and they usually have antibodies to p40tax. Moreover, a study of 250 randomly selected blood donors revealed that approximately 8% of these seronegative individuals also had HTLV-1 tax sequences and antibodies to p40tax, while they lacked sequences and antibodies related to gag, pol, or env. Thus, it seemed important to determine whether the “tax-only” state can be transmitted by transfusion. To this end, PBMCs from HTLV-1 and -2 seronegativetax-only-positive MF patients or from healthytax-only-positive blood donors were injected into adult rabbits, an established animal model for HTLV-1 infection. The PBMCs of all injected rabbits became tax sequence positive. These observations suggest that HTLV-1 tax can be transmitted bytax-only-positive mononuclear cells.

White cell reduction by filtration may significantly decrease human T− lymphotropic virus type 1 Tax sequences and Tax‐encoded proteins in blood used for transfusion

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