Hypomorphic RAG deficiency: impact of disease burden on survival and thymic recovery argues for early diagnosis and HSCT.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Immunotherapy with gene-modified T cells: limiting side effects provides new challenges.

Genetic tools have been developed to efficiently engineer T-cell specificity and enhance T-cell function. Chimeric antigen receptors (CAR) use the antibody variable segments to direct specificity against cell surface molecules. T-cell receptors (TCR) can redirect T cells to intracellular target proteins, fragments of which are presented in the peptide-binding groove of HLA molecules. A recent clinical trial with CAR-modified T cells redirected against the B-cell lineage antigen CD19 showed dramatic clinical benefit in chronic lymphocytic leukaemia patients. Similarly, impressive clinical responses were seen in melanoma and synovial cell carcinoma with TCR-modified T cells redirected against the melanocyte lineage antigen MART-1 and the testis-cancer antigen NY-ESO-1. However, on and off-target toxicity was associated with most of these clinical responses, and fatal complications have been observed in some patients treated with gene modified T cells. This review will discuss factors that might contribute to toxic side effects of therapy with gene modified T cells, and outline potential strategies to retain anticancer activity while reducing unwanted side effects.

Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

Allogeneic hematopoietic stem cell transplantation and norovirus gastroenteritis: a previously unrecognized cause of morbidity.

BACKGROUND: A retrospective study of the clinical, epidemiologic, and virologic features of norovirus gastroenteritis in 12 adult allogeneic hematopoietic stem cell transplant (HSCT) recipients. METHODS: Norovirus infection was diagnosed by reverse-transcriptase polymerase chain reaction. Strains were genotyped by nucleic acid sequence of the most highly conserved region of the norovirus gene encoding the capsid S (shell) domain. RESULTS: Ten of 12 patients presented with vomiting of short duration, but diarrhea was present in all. The median time from onset to norovirus diagnosis was 1 month (range, 0.25-6.0 months). Eleven patients were receiving immunosuppression when norovirus infection was diagnosed: 8 for graft-versus-host disease (GVHD) in an organ other than gut, 1 for previous gut GVHD, and 2 for presumed gut GVHD that proved to be norovirus gastroenteritis. Six patients required enteral or parenteral nutrition for severe weight loss. In 10 patients, diarrhea lasted a median of 3 months (range, 0.5-14 months) and virus was shed at a high level throughout. The remaining 2 patients died after 4 months of diarrhea (one died of unrelated complications, and the other died of malnutrition). The noroviruses found were GII (untyped), GII-3, GII-4, and GII-7 in 1, 1, 9, and 1 patients, respectively. Eleven of the 12 patients had acquired their infection in the community. Phylogenetic analysis of the GII-4 strains demonstrated that all differed. CONCLUSIONS: Noroviruses are a hitherto unsuspected cause of prolonged morbidity and mortality in adults after allogeneic HSCT. The use of reverse-transcriptase polymerase chain reaction to detect high viral load levels in feces distinguishes norovirus gastroenteritis from gut GVHD.

Retroviral transfer of a dominant TCR prevents surface expression of a large proportion of the endogenous TCR repertoire in human T cells.

The latent membrane protein-2 (LMP2) of Epstein-Barr virus is a potential target for T-cell receptor (TCR) gene therapy of Hodgkin lymphoma and nasopharyngeal carcinoma. Here, we modified a human leukocyte antigen-A2-restricted, LMP2-specific TCR to achieve efficient expression following retroviral TCR gene transfer. The unmodified TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. In order to improve this TCR, we replaced the human constant region with murine sequences, linked the two TCR genes using a self-cleaving 2A sequence and finally, codon optimized the TCR-alpha-2A-beta cassette for efficient translation in human cells. Retroviral transfer of the modified TCR resulted in efficient surface expression and HLA-A2/LMP2 pentamer binding. The transduced cells showed peptide-specific interferon-gamma and interleukin-2 production and killed target cells displaying the LMP2 peptide. Importantly, the introduced LMP2-TCR suppressed the cell surface expression of a large proportion of endogenous TCR combinations present in primary human T cells. The design of dominant TCR is likely to improve TCR gene therapy by reducing the risk of potential autoreactivity of endogenous and mispaired TCR combinations.

Superiority of reduced-intensity allogeneic transplantation over conventional treatment for relapse of Hodgkin's lymphoma following autologous stem cell transplantation.

This study compares outcome of reduced-intensity conditioned transplant (RIT) with outcome of conventional non-transplant therapy in patients with Hodgkin's lymphoma relapsing following autograft. There were 72 patients in two groups who had relapsed, and received salvage therapy with chemotherapy+/-radiotherapy. One group (n=38) then underwent alemtuzumab-containing RIT. The second group-historical controls (n=34), relapsing before the advent of RIT-had no further high-dose therapy. This group was required to respond to salvage therapy and live for over 12 months post-relapse, demonstrating potential eligibility for RIT, had this been available. Overall survival (OS) from diagnosis was superior following RIT (48% at 10 years versus 15%; P=0.0014), as was survival from autograft (65% at 5 years versus 15%; P< or =0.0001). For the RIT group, OS at 5 years from allograft was 51%, and in chemoresponsive patients was 58%, with current progression-free survival of 42%. Responses were seen in 8 of 15 patients receiving donor lymphocyte infusions (DLI) for relapse/progression, with durable remission in five patients at median follow-up from DLI of 45 months (28-55). These data demonstrate the potential efficacy of RIT in heavily pre-treated patients whose outlook with conventional therapy is dismal, and provide evidence of a clinically relevant graft-versus-lymphoma effect.

Investigations into seed dormancy in Grevillea linearifolia, G. buxifolia and G. sericea: anatomy and histochemistry of the seed coat.

BACKGROUND AND AIMS: Seeds of east Australian Grevillea species generally recruit post-fire; previous work showed that the seed coat was the controller of dormancy in Grevillea linearifolia. Former studies on seed development in Grevillea have concentrated on embryology, with little information that would allow testing of hypotheses about the breaking of dormancy by fire-related cues. Our aim was to investigate structural and chemical characteristics of the seed coat that may be related to dormancy for three Grevillea species. METHODS: Seeds of Grevillea linearifolia, Grevillea buxifolia and Grevillea sericea were investigated using gross dissection, thin sectioning and histochemical staining. Water movement across the seed coat was tested for by determining the water content of embryos from imbibed and dry seeds of G. sericea. Penetration of intact seeds by Lucifer Yellow was used to test for internal barriers to diffusion of high-molecular-weight compounds. KEY RESULTS: Two integuments were present in the seed coat: an outer testa, with exo-, meso- and endotestal (palisade) layers, and an inner tegmen of unlignified sclerenchyma. A hypostase at the chalazal end was a region of structural difference in the seed coat, and differed slightly among the three species. An internal cuticle was found on each side of the sclerenchyma layer. The embryos of imbibed seeds had a water content six times that of dry seeds. Barriers to diffusion of Lucifer Yellow existed at the exotestal and the endotestal/hypostase layers. CONCLUSIONS: Several potential mechanisms of seed coat dormancy were identified. The embryo appeared to be completely surrounded by outer and inner barriers to diffusion of high-molecular-weight compounds. Phenolic compounds present in the exotesta could interfere with gas exchange. The sclerenchyma layer, together with strengthening in the endotestal and exotestal cells, could act as a mechanical constraint.

Use of 18F-FDG positron emission tomography following allogeneic transplantation to guide adoptive immunotherapy with donor lymphocyte infusions.

Fluorine-18 fluorodeoxyglucose positron emission tomography ((18)F-FDG PET) provides valuable prognostic information in the management of lymphoma patients. However, the utility of (18)F-FDG PET following allografting is unclear. We analysed the use of (18)F-FDG PET after allogeneic reduced-intensity transplantation (RIT) performed in our institution. Between June 1998 and January 2002, 55 patients underwent RIT for either Hodgkin or non-Hodgkin lymphoma. At least one (18)F-FDG PET scan was performed during the post-transplant period (median five studies) in 15 (27.2%) of these 55 patients. PET scans were performed after re-staging computed tomography (CT) and were categorised depending on (18)F-FDG uptake. The first PET scan was informative in 11 of 15 patients (73%) and influenced the administration of donor lymphocyte infusions (DLI) in nine: leading to earlier DLI administration in two patients, earlier dose escalation in one, withholding of DLI administration in five and dose reduction in one. In addition, subsequent monitoring with (18)F-FDG PET scans documented a graft-versus-lymphoma effect in five patients (median post-DLI follow-up 33 months, range 13-36 months). These preliminary data suggest that (18)F-FDG PET has a role in guiding DLI administration and monitoring the immunotherapeutic effect in patients after allogeneic transplantation. This retrospective pilot study forms the basis for a prospective study to clarify the utility of (18)F-FDG PET/CT in these patients.

Prospects for immunotherapy of malignant disease.

The majority of T cell-recognized tumour antigens in humans are encoded by genes that are also present in normal tissues. Low levels of gene expression in normal cells can lead to the inactivation of high-avidity T cells by immunological tolerance mechanisms. As a consequence, low-avidity T cell responses in patients are often inadequate in providing tumour protection. Recently, several technologies have been developed to overcome tolerance, allowing the isolation of high-affinity, HLA-restricted receptors specific for tumour-associated peptide epitopes. Furthermore, transfer of HLA-restricted antigen receptors provides an opportunity to empower patient T cells with new tumour-reactive specificities that cannot be retrieved from the autologous T cell repertoire.

Environmental exposure to cockroach allergens: analysis with monoclonal antibody-based enzyme immunoassays.

Quantitative two-site monoclonal antibody (MAb)-based enzyme-linked immunoassays for two cockroach (CR) allergens, Bla g I and Bla g II, have been developed and used to measure allergen levels in house-dust samples. Dust collected from the CR-infested homes of two patients with asthma from Charlottesville, Va., demonstrated wide variation in the levels of Bla g I, depending on the location of dust collection. Dust from kitchen floors and cabinets contained 50-fold more allergen (mean, 10,755 U/gm of dust) than dust from bedrooms and upholstered furniture (mean, 204 U/gm). One hundred forty-five dust samples were collected from the bedrooms and living rooms of 22 children with asthma and 16 control subjects without asthma living in Atlanta, Ga. Twenty-seven of the 38 homes (17/22 children with asthma; 10/16 control subjects) had detectable Bla g I (4 to 1340 U/gm of dust). Bla g II levels were assayed in 40 kitchen, bedroom, and living room samples from homes in Wilmington, Del. Highest levels of Bla g II were detected in kitchen-floor dust (300 U/gm of dust). Additionally, approximately 20% of homes with no visual evidence of CR infestation had significant levels of Bla g II in at least one dust sample (greater than 4 U/gm of dust). Our results demonstrate that CR may be an occult allergen in homes. The kitchen appears to be the primary site of CR-allergen accumulation, but significant CR-allergen levels can also be found at other sites in the home. The MAb-based assays can be used for quantitation of environmental exposure to CR allergens.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacotherapy of allergic disease.

In 1987 the clinician has available to him a wide range of pharmacologic agents with which to treat allergic disease. These drugs used alone or in combination may eliminate or significantly modify allergic symptoms in any organ system. The appropriate use of several drugs in combination can alleviate the need to accept partial resolution of symptoms. However, due to the complexity of some of these agents and the possibility of drug interactions and associated side effects a thorough understanding of their use and pharmacology is needed.

IgG subclasses in nonallergic children with chronic chest symptoms.

Immunoglobulin and IgG subclass measurements were made on sera from 37 children thought to have asthma whose chronic chest symptoms were unexplained by allergy. There was a higher proportion of low or low-normal levels of IgG subclasses 1, 2, and 4 in these children than in normal children. Those who had low serum IgG values on initial measurement had a higher proportion of low or low-normal levels of IgG1, IgG2, and IgG4; those who had normal IgG values had a higher proportion of low or low-normal levels of IgG2 and IgG4. Thus a normal serum concentration of IgG did not exclude the possibility of an abnormal level of IgG2 or IgG4. Our experience suggests that abnormal levels of IgG subclasses might play an etiologic role in the chronic chest symptoms in some of these children.

The surface of Mars: the view from the viking 2 lander.

Viking 2 lander began imaging the surface of Mars at Utopia Planitia on 3 September 1976. The surface is a boulder-strewn reddish desert cut by troughs that probably form a polygonal network. A plateau can be seen to the east of the spacecraft, which for the most probable lander location is approximately the direction of a tongue of ejecta from the crater Mie. Boulders at the lander 2 site are generally more vesicular than those near lander i. Fines at both lander sites appear to be very fine-grained and to be bound in a duricrust. The pinkish color of the sky, similar to that observed at the lander I site, indicates suspension of surface material. However, the atmospheric optical depth is less than that at the lander I site. After dissipation of a cloud of dust stirred during landing, no changes other than those stemming from sampling activities have been detected in the landscape. No signs of large organisms are apparent at either landing site.