Extraordinary clinical response to ibrutinib in low-grade ovarian cancer guided by organoid drug testing.

Low-grade serous ovarian cancer (LGSOC) typically responds poorly to standard platinum-based chemotherapy and new therapeutic approaches are needed. We describe a remarkable response to targeted therapy in a patient with platinum-resistant, advanced LGSOC who had failed standard-of-care chemotherapy and two surgeries. The patient was in rapid decline and entering hospice care on home intravenous (i.v.) opioid analgesics and a malignant bowel obstruction requiring a G-tube. Genomic analysis of the patient's tumor did not indicate obvious therapeutic options. In contrast, a CLIA-certified drug sensitivity assay of an organoid culture derived from the patient's tumor identified several therapeutic choices, including Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, as well as the EGFR inhibitors afatinib and erlotinib. Following off-label administration of daily ibrutinib as monotherapy, the patient had an exceptional clinical turnaround over the following 65 weeks with normalization of CA-125 levels, resolution of the malignant bowel obstruction, halting of pain medications, and improvement of performance status from ECOG 3 to ECOG 1. After 65 weeks of stable disease, the patient's CA-125 levels began to rise, at which point the patient discontinued ibrutinib and began taking afatinib as monotherapy. The patient's CA-125 levels remained stable for an additional 38 weeks but due to anemia and rising CA-125 levels, the patient switched to erlotinib and is currently being monitored. This case highlights the clinical utility of ex vivo drug testing of patient-derived tumor organoids as a new functional precision medicine approach to identify effective personalized therapies for patients who have failed standard-of-care treatments.

Case report: ex vivo tumor organoid drug testing identifies therapeutic options for stage IV ovarian carcinoma.

Patients presenting with stage 4 ovarian carcinoma, including low-grade serous disease, have a poor prognosis. Although platinum-based therapies can offer some response, these therapies are associated with many side effects, and treatment resistance often develops. Toxic side effects along with disease progression render patients unable to receive additional lines of treatment and limit their options to hospice or palliative care. In this case report, we describe a patient with an unusual case of metastatic low-grade serous ovarian cancer with some features of high-grade disease who had received four previous lines of treatment and was suffering from atelectasis, pulmonary embolism, and hydronephrosis. A CLIA-certified drug sensitivity assay of an organoid culture derived from the patient's tumor (PARIS® test) identified several therapeutic options, including the combination of fulvestrant with everolimus. On this treatment regimen, the patient experienced 7 months of stable disease and survived nearly 11 months before succumbing to her disease. This case emphasizes the clinical utility of ex vivo drug testing as a new functional precision medicine approach to identify, in real-time, personalized treatment options for patients, especially those who are not benefiting from standard of care treatments.

Medium-throughput Drug Screening of Patient-derived Organoids from Colorectal Peritoneal Metastases to Direct Personalized Therapy.

PURPOSE: Patients with colorectal cancer with peritoneal metastases (CRPMs) have limited treatment options and the lowest colorectal cancer survival rates. We aimed to determine whether organoid testing could help guide precision treatment for patients with CRPMs, as the clinical utility of prospective, functional drug screening including nonstandard agents is unknown. EXPERIMENTAL DESIGN: CRPM organoids (peritonoids) isolated from patients underwent parallel next-generation sequencing and medium-throughput drug panel testing ex vivo to identify specific drug sensitivities for each patient. We measured the utility of such a service including: success of peritonoid generation, time to cultivate peritonoids, reproducibility of the medium-throughput drug testing, and documented changes to clinical therapy as a result of the testing. RESULTS: Peritonoids were successfully generated and validated from 68% (19/28) of patients undergoing standard care. Genomic and drug profiling was completed within 8 weeks and a formal report ranking drug sensitivities was provided to the medical oncology team upon failure of standard care treatment. This resulted in a treatment change for two patients, one of whom had a partial response despite previously progressing on multiple rounds of standard care chemotherapy. The barrier to implementing this technology in Australia is the need for drug access and funding for off-label indications. CONCLUSIONS: Our approach is feasible, reproducible, and can guide novel therapeutic choices in this poor prognosis cohort, where new treatment options are urgently needed. This platform is relevant to many solid organ malignancies.

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity.

BACKGROUND AIMS: Key obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells. METHODS: We delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3+Vα24+Vß11+ iNKT cells. RESULTS: Gene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4+ and CD8+ responder T cells. DISCUSSION: These data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.

Comparison of sequence-specific oligonucleotide probe vs next generation sequencing for HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single-pass high-resolution HLA typing in support of solid organ and hematopoietic cell transplant programs.

Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the "gold standard." However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation.

An integrated genotyping approach for HLA and other complex genetic systems.

Clinical immunogenetics laboratories performing routine sequencing of human leukocyte antigen (HLA) genes in support of hematopoietic cell transplantation are motivated to upgrade to next-generation sequencing (NGS) technology by its potential for cost savings as well as testing accuracy and flexibility. While NGS machines are available and simple to operate, there are few systems available that provide comprehensive sample preparation and data analysis workflows to complete the process. We report on the development and testing of the Integrated Genotyping System (IGS), which has been designed to specifically address the challenges associated with the adoption of NGS in clinical laboratories. To validate the system for a variety of sample DNA sources, we have tested 336 DNA specimens from whole blood, dried blood spots, buccal swabs, and lymphoblastoid cell lines. HLA class I and class II genotypes were derived from amplicon sequencing of HLA-A, -B, -C for exons 1-7 and HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, -DRB5 for exons 1-4. Additionally, to demonstrate the extensibility of the IGS to other genetic loci, KIR haplotyping of 93 samples was carried out in parallel with HLA typing using a workflow based on the HLA system. These results are discussed with respect to their applications in the clinical setting and consequent potential for advancing precision medicine.

Next generation sequencing to determine HLA class II genotypes in a cohort of hematopoietic cell transplant patients and donors.

Current high-resolution HLA typing technologies frequently produce ambiguous results that mandate extended testing prior to reporting. Through multiplex sequencing of individual amplicons from many individuals at multiple loci, next generation sequencing (NGS) promises to eliminate heterozygote ambiguities and extend the breadth of genetic data acquired with little additional effort. We report here on assessment of a novel NGS HLA genotyping system for resequencing exons 2 and 3 of DRB1/B3/B4/B5, DQA1 and DQB1 and exon 2 of DPA1 and DPB1 on the MiSeq platform. In a cohort of 2605 hematopoietic cell transplant recipients and donors, NGS achieved 99.6% accuracy for DRB1 allele assignments and 99.5% for DQB1, compared to legacy genotypes generated pretransplant. NGS provided at least single 4-digit allele resolution for 97% of genotypes at DRB1 and 100% at DQB1. Overall, NGS typing identified 166 class II alleles, including 9 novel sequences with greater than 99% accuracy for DRB1 and DQB1 genotypes and elimination of diploid ambiguities through in-phase sequencing demonstrated the robust reliability of the NGS HLA genotyping reagents and analysis software employed in this study.

Cytomegalovirus-specific T cells are primed early after cord blood transplant but fail to control virus in vivo.

A disadvantage of umbilical cord blood transplantation (UCBT) is the delay in immune reconstitution, placing patients at increased risk for infections after transplant. Cytomegalovirus (CMV) in particular has been shown to cause significant morbidity in patients undergoing UCBT. Here, we comprehensively evaluate the development of CD4(+) and CD8(+) T-cell responses to CMV in a cohort of patients that underwent double UCBT. Our findings demonstrate conclusively that a diverse polyclonal CMV-specific T-cell response derived from the UCB graft is primed to viral antigens as early as day 42 after UCBT, but these T cells fail to achieve sufficient numbers in vivo to control CMV reactivations. This is not due to an inherent inability of UCB-derived T cells to proliferate, as these T cells underwent rapid proliferation in vitro. The TCR diversity and antigen specificity of CMV-specific T cells remained remarkably stable in the first year after transplant, suggesting that later control of virus replication results from improved function of T cells primed early after transplant and not from de novo responses derived from later thymic emigrants. Ex vivo expansion and adoptive transfer of CMV-specific T cells isolated from UCBT recipients early after transplant could augment immunity to CMV.

Transfusion-associated graft-versus-host disease in a liver transplant recipient: an unusual presentation and review of the literature.

BACKGROUND: Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare, nearly universally fatal complication from transfusion of nonirradiated cellular blood components, occurring when a recipient's immune system is unable to recognize and destroy transfused T lymphocytes. Irradiation of cellular components eliminates this risk. We present an unusual case of a liver transplant recipient developing TA-GVHD 13 weeks after transfusion of a random unit of nonirradiated red blood cells (RBCs) that happened to be from a donor homozygous for an HLA haplotype shared by the patient. STUDY DESIGN AND METHODS: This study was a single case review of a liver transplant recipient who developed skin GVHD and marrow aplasia. Clinical course and the chimerism studies involving the patient, the liver donor, and the blood donor are detailed. RESULTS: The patient presented 3 months posttransplant with GVHD of his skin and marrow aplasia. In addition to standard antigraft immunosuppression, this patient had started the interleukin-1 receptor antagonist anakinra on Posttransplant Day 13 for an acute gout flare. Chimerism studies on the patient's peripheral blood identified a population of CD3 cells that did not originate with either the patient or his liver donor. HLA studies and microsatellite profiling of the unknown CD3 population identified the source of the patient's TA-GVHD, a unit of nonirradiated, nonleukoreduced apheresis RBCs. CONCLUSION: Use of an immunomodulating agent may have contributed to the development of TA-GVHD in a liver transplant patient who received a random unit of nonirradiated RBCs by chance from an unrelated haploidentical donor.

Short tandem repeat and human leukocyte antigen mutations or losses confound engraftment and typing analysis in hematopoietic stem cell transplants.

Clonal chromosomal abnormalities are often found in the tumor cells of patients with malignancies. These abnormalities can cause downregulation of human leukocyte antigen (HLA) and instability of short tandem repeat (STR) DNA sequences, confounding HLA typing and/or engraftment analysis in hematopoietic stem cell transplants (HSCT). We describe here the abnormalities observed during testing of 600 HSCT patients. HLA molecular typing was performed by reference strand conformational analyses and/or sequence-based typing. STR testing was performed with 10 to 16 STR primer sets, following 1 to 4 informative loci in each patient. Eight patients exhibited either loss of heterozygosity (4 STR, 3 HLA) or STR length mutation (n = 1), and 5 of the 8 exhibited correlative cytogenetic abnormalities. Diagnoses were acute myelogenous leukemia (AML; n = 7) or myelofibrosis (MFIB: n = 1), yielding an 11% incidence of these chromosomal abnormalities among the subset of 72 AML/MFIB HSCT patients. These results highlight some of the problems encountered and the possibility for interpretive errors that can arise when analyzing molecular typing and engraftment data, particularly among AML/MFIB patients.

Donor-specific antibody against denatured HLA-A1: clinically nonsignificant?

Pre-transplant screening of a woman with end-stage renal disease (ESRD) showed no anti-human leukocyte antigen (HLA) alloantibodies by anti-human globulin-complement-dependent cytotoxicity (AHG-CDC; class I) or enzyme-linked immunosorbent assay (class II). Following a negative AHG-CDC crossmatch, an HLA*01:01+ deceased donor (DD) kidney was transplanted in September 2005. Subsequent screening of pre-transplant serum by LABScreen Single Antigen (SA) array showed strong reactivity versus A*01:01. Despite that reactivity, at 5 years post-transplant, the patient has a serum creatinine of 1.6 mg/dl and has never experienced humoral or cellular rejection. Retrospective flow-cytometric crossmatch of pre- and post-transplant sera versus DD cells was negative. Rescreening of multiple pre- and post-transplant sera revealed anti-A1 reactivity persisting from the first through the last samples tested. The patient's anti-A1 was almost two fold more reactive with denatured A*01:01 FlowPRA SA beads after denaturation with acid treatment (pH 2.7) than with untreated beads. Parallel results were observed with pH 2.7 treated versus untreated A1+ T cells in FXM. These data highlight the difficulty in interpreting screening results obtained using bead arrays, because of antibodies that appear to recognize denatured but not native class I HLA antigens. We suggest that such bead-positive, flow cytometric crossmatch negative antibodies are not associated with humoral rejection, may not necessarily be detrimental to a graft, and deserve further evaluation before becoming a barrier to transplantation.

Comparative analysis of risk factors for acute graft-versus-host disease and for chronic graft-versus-host disease according to National Institutes of Health consensus criteria.

Risk factors for grades 2-4 acute graft-versus-host disease (GVHD) and for chronic GVHD as defined by National Institutes of Health consensus criteria were evaluated and compared in 2941 recipients of first allogeneic hematopoietic cell transplantation at our center. In multivariate analyses, the profiles of risk factors for acute and chronic GVHD were similar, with some notable differences. Recipient human leukocyte antigen (HLA) mismatching and the use of unrelated donors had a greater effect on the risk of acute GVHD than on chronic GVHD, whereas the use of female donors for male recipients had a greater effect on the risk of chronic GVHD than on acute GVHD. Total body irradiation was strongly associated with acute GVHD, but had no statistically significant association with chronic GVHD, whereas grafting with mobilized blood cells was strongly associated with chronic GVHD but not with acute GVHD. Older patient age was associated with chronic GVHD, but had no effect on acute GVHD. For all risk factors associated with chronic GVHD, point estimates and confidence intervals were not significantly changed after adjustment for prior acute GVHD. These results suggest that the mechanisms involved in acute and chronic GVHD are not entirely congruent and that chronic GVHD is not simply the end stage of acute GVHD.

Multiple roles of Lyn kinase in myeloid cell signaling and function.

Lyn is an Src family kinase present in B lymphocytes and myeloid cells. In these cell types, Lyn establishes signaling thresholds by acting as both a positive and a negative modulator of a variety of signaling responses and effector functions. Lyn deficiency in mice results in the development of myeloproliferation and autoimmunity. The latter has been attributed to the hyper-reactivity of Lyn-deficient B cells due to the unique role of Lyn in downmodulating B-cell receptor activation, mainly through phosphorylation of inhibitory molecules and receptors. Myeloproliferation results, on the other hand, from the enhanced sensitivity of Lyn-deficient progenitors to a number of colony-stimulating factors (CSFs). The hyper-sensitivity to myeloid growth factors may also be secondary to poor inhibitory receptor phosphorylation, leading to impaired recruitment/activation of tyrosine phosphatases and reduced downmodulation of CSF signaling responses. Despite these observations, the overall role of Lyn in the modulation of myeloid cell effector functions is much less well understood, as often both positive and negative roles of this kinase have been reported. In this review, we discuss the current knowledge of the duplicitous nature of Lyn in the modulation of myeloid cell signaling and function.

Prospective monitoring for alloimmunization in cord blood transplantation: "virtual crossmatch" can be used to demonstrate donor-directed antibodies.

Preformed host antibodies may contribute to graft rejection after hematopoietic stem-cell transplantation. In cord blood transplantation (CBT), donor-directed host antibodies may be particularly relevant because patients are often markedly mismatched to donors, and limited donor cells preclude cross-matching. The recent development of single human leukocyte antigen (HLA) microbead array assays allows characterization of host alloreactivity to individual HLA antigens with sufficient sensitivity and specificity to allow consideration of "virtual crossmatch" testing as a surrogate for conventional crossmatch testing in the CBT setting. We report results of prospective monitoring for alloimmunization in our recent CBT experience. Among 46 consecutive patients, four patients (9%) (5 of 88 units [6%]) had evidence of at least moderate antibodies to HLA antigens on cord units originally selected for transplantation. Virtual crossmatch can be used to screen for donor-directed antibodies in CBT. As possible, units should be changed to avoid sensitized mismatches.

Neonatal alloimmune thrombocytopenia and neutropenia associated with maternal human leukocyte antigen antibodies.

Neonatal thrombocytopenia or neutropenia may result from passive transfusion of maternally derived antibodies. Antibodies against platelet antigens are commonly associated with neonatal alloimmune thrombocytopenia (NAIT), and anti-neutrophil antibodies are frequently identified in alloimmune neonatal neutropenia (ANN). Combined alloimmune cytopenias in the newborn are rarely reported; even fewer reports document human leukocyte antigen (HLA) antibodies as a potential cause of neonatal thrombocytopenia or neutropenia. We describe neutropenia and thrombocytopenia in a newborn associated with markedly elevated maternal HLA antibodies in the absence of anti-neutrophil or anti-platelet antibodies to highlight consideration of HLA antibodies in the pathogenesis of ANN and NAIT.

H-Y antibody development associates with acute rejection in female patients with male kidney transplants.

BACKGROUND: Human minor histocompatibility antigens (mHA) and clinically relevant immune responses to them have not been well defined in organ transplantation. We hypothesized that women with male kidney transplants would develop antibodies against H-Y, the mHA encoded on the Y-chromosome, in association with graft rejection. METHODS: We tested sera from 118 consecutive transplant recipients with kidney biopsies. Antibodies that specifically recognized the recombinant H-Y antigens RPS4Y1 or DDX3Y were detected by IgG enzyme-linked immunosorbent assay and western blotting. Immunogenic epitopes were further identified using overlapping H-Y antigen peptides for both the H-Y proteins. RESULTS: In the 26 female recipients of male kidneys, H-Y antibody development posttransplant (1) was more frequent (46%) than in other gender combinations (P<0.001), (2) showed strong correlation with acute rejection (P=0.00048), (3) correlated with plasma cell infiltrates in biopsied kidneys (P=0.04), and (4) did not correlate with C4d deposition or donor-specific anti-human leukocyte antigen (HLA) antibodies. Of the two H-Y antigens, RPS4Y1 was more frequently recognized (P=0.005). CONCLUSION: This first demonstration of a strong association between H-Y antibody development and acute rejection in kidney transplant recipients shows that in solid organ allografts, humoral immune responses against well defined mHA have clear clinical correlates, can be easily monitored, and warrant study for possible effects on long-term graft function.

Src family kinases are important negative regulators of G-CSF-dependent granulopoiesis.

Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis. Truncation mutations of the G-CSF receptor (G-CSFR) are associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia. Although increased proliferative signaling by a representative G-CSFR truncation mutation (termed d715) has been documented, the molecular basis for this hyperproliferative phenotype has not been fully characterized. Given the accumulating evidence implicating Src family kinases in the transduction of cytokine receptor signals, the role of these kinases in the regulation of G-CSF signaling was examined. We show that Hck and Lyn, Src family kinases expressed in myeloid cells, are negative regulators of granulopoiesis that act at distinct stages of granulocytic differentiation. Whereas Hck regulates the G-CSF-induced proliferation of granulocytic precursors, Lyn regulates the production of myeloid progenitors. Interestingly, d715 G-CSFR myeloid progenitors were resistant to the growth-stimulating effect of treatment with a Src kinase inhibitor. Together, these data establish Lyn and Hck as key negative regulators of granulopoiesis and raise the possibility that loss of Src family kinase activation by the d715 G-CSFR may contribute to its hyperproliferative phenotype.

The inhibitory receptor PIR-B negatively regulates neutrophil and macrophage integrin signaling.

The Ig-like receptor family member, PIR-B, has been shown to play an inhibitory role in receptor signaling within B cells, mast cells, and dendritic cells. As it has been implicated in integrin-mediated responses, we investigated the effect of loss of the PIR-B protein on integrin-mediated signaling in primary murine myeloid cells. The pir-b-/- neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when plated on surfaces coated with either extracellular matrix proteins or cellular adhesion molecules in the presence or absence of the soluble inflammatory agonist TNF-alpha. The pir-b-/- and wild-type cells responded equivalently when stimulated with TNF-alpha in suspension, indicating that the hyperresponsive phenotype of the pir-b-/- cells during adhesion was due to enhanced integrin signaling. Both wild-type and pir-b-/- neutrophils expressed similar levels of integrin subunits. Primary bone marrow-derived macrophages from pir-b-/- mice were also hyperadhesive and spread more rapidly than wild-type cells following plating on surfaces that cross-linked cellular beta2 integrins. Biochemical analysis of macrophages from pir-b-/- mice revealed enhanced phosphorylation and activation of proteins involved in integrin signaling. These observations point to a nonredundant role for PIR-B in the regulation of leukocyte integrin signaling.

The Lyn tyrosine kinase negatively regulates neutrophil integrin signaling.

The Src family kinase Lyn has been shown to play both stimulatory and inhibitory roles within several hemopoietic cell types. In this study, we investigated the role played by Lyn in neutrophil integrin signaling. Loss of Lyn resulted in a hyperresponsive phenotype on engagement of surface integrins at low valency. Lyn(-/-) neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when adherent to surfaces coated with either cellular counterreceptors or extracellular matrix proteins. In contrast, Lyn-deficient and wild-type cells expressed similar levels of surface integrins and responded equivalently to activating agents in suspension, indicating that the enhanced responses of lyn(-/-) cells was specific to the integrin signaling pathways. Lyn-deficient macrophages also displayed a hyperadhesive phenotype. Biochemical analysis of macrophages from lyn(-/-) mice revealed that Lyn plays an essential role in the adhesion-dependent phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of the inhibitory receptors SIRP1alpha and PIR-B, which in turn recruit the phosphatase SHP-1. These observations suggest that reduced mobilization of SHP-1 to the membrane in lyn(-/-) neutrophils results in a hyperadhesive and hyperactive phenotype. This hypothesis is further supported by the fact that neutrophils from me(v)/me(v) mice, which have significantly reduced SHP-1 activity, are also hyperresponsive following integrin engagement. This is the first direct evidence using primary leukocytes from lyn(-/-) mice that this kinase functions as a negative regulator in integrin signaling.

Rough eye is a gain-of-function allele of amos that disrupts regulation of the proneural gene atonal during Drosophila retinal differentiation.

The regular organization of the ommatidial lattice in the Drosophila eye originates in the precise regulation of the proneural gene atonal (ato), which is responsible for the specification of the ommatidial founder cells R8. Here we show that Rough eye (Roi), a dominant mutation manifested by severe roughening of the adult eye surface, causes defects in ommatidial assembly and ommatidial spacing. The ommatidial spacing defect can be ascribed to the irregular distribution of R8 cells caused by a disruption of the patterning of ato expression. Disruptions in the recruitment of other photoreceptors and excess Hedgehog production in differentiating cells may further contribute to the defects in ommatidial assembly. Our molecular characterization of the Roi locus demonstrates that it is a gain-of-function mutation of the bHLH gene amos that results from a chromosomal inversion. We show that Roi can rescue the retinal developmental defect of ato1 mutants and speculate that amos substitutes for some of ato's function in the eye or activates a residual function of the ato1 allele.