Generating real-world data from health records: design of a patient-centric study in multiple sclerosis using a commercial health records platform.

OBJECTIVE: The FlywheelMS study will explore the use of a real-world health record data set generated by PicnicHealth, a patient-centric health records platform, to improve understanding of disease course and patterns of care for patients with multiple sclerosis (MS). MATERIALS AND METHODS: The FlywheelMS study aims to enroll 5000 adults with MS in the United States to create a large, deidentified, longitudinal data set for clinical research. PicnicHealth obtains health records, including paper charts, electronic health records, and radiology imaging files from any healthcare site. Using a large-scale health record processing pipeline, PicnicHealth abstracts standard and condition-specific data elements from structured (eg, laboratory test results) and unstructured (eg, narrative) text and maps these to standardized medical vocabularies. Researchers can use the resulting data set to answer empirical questions and study participants can access and share their harmonized health records using PicnicHealth's web application. RESULTS: As of November 24, 2020, more than 4176 participants from 49 of 50 US states have enrolled in the FlywheelMS study. A median of 200 pages of records have been collected from 14 different doctors over 8 years per participant. Abstraction precision, established through inter-abstractor agreement, is as high as 97.8% when identifying and mapping data elements to a standard ontology. CONCLUSION: Using a commercial health records platform, the FlywheelMS study is generating a real-world, multimodal data set that could provide valuable insights about patients with MS. This approach to data collection and abstraction is disease-agnostic and could be used to address other clinical research questions in the future.

Increased potency of recombinant VWF D'D3 albumin fusion proteins engineered for enhanced affinity for coagulation factor VIII.

BACKGROUND: We have recently reported on a recombinant von Willebrand factor (VWF) D'D3 albumin fusion protein (rD'D3-FP) developed to extend the half-life of coagulation factor VIII (FVIII) for the treatment of hemophilia A. Based on predictive modelling presented in this study, we hypothesized that modifying rD'D3-FP to improve FVIII interaction would reduce exchange with endogenous VWF and provide additional FVIII half-life benefit. OBJECTIVES: The aim of this study was to identify novel rD'D3-FP variants with enhanced therapeutic efficacy in extending FVIII half-life. METHODS: Through both directed mutagenesis and random mutagenesis using a novel mammalian display platform, we identified novel rD'D3-FP variants with increased affinity for FVIII (rVIII-SingleChain) under both neutral and acidic conditions and assessed their ability to extend FVIII half-life in vitro and in vivo. RESULTS: In rat preclinical studies, rD'D3-FP variants with increased affinity for FVIII displayed enhanced potency, with reduced dose levels required to achieve equivalent rVIII-SingleChain half-life extension. In cell-based imaging studies in vitro, we also demonstrated reduced dissociation of rVIII-SingleChain from the rD'D3-FP variants within acidic endosomes and more efficient co-recycling of the rD'D3-FP/rVIII-SingleChain complex via the FcRn recycling system. CONCLUSIONS: In summary, at potential clinical doses, the rD'D3-FP variants provide marked benefits with respect to dose levels and half-life extension of co-administered FVIII, supporting their development for use in the treatment of hemophilia A.

Prognostic model to identify and quantify risk factors for mortality among hospitalised patients with COVID-19 in the USA.

OBJECTIVES: To develop a prognostic model to identify and quantify risk factors for mortality among patients admitted to the hospital with COVID-19. DESIGN: Retrospective cohort study. Patients were randomly assigned to either training (80%) or test (20%) sets. The training set was used to fit a multivariable logistic regression. Predictors were ranked using variable importance metrics. Models were assessed by C-indices, Brier scores and calibration plots in the test set. SETTING: Optum de-identified COVID-19 Electronic Health Record dataset including over 700 hospitals and 7000 clinics in the USA. PARTICIPANTS: 17 086 patients hospitalised with COVID-19 between 20 February 2020 and 5 June 2020. MAIN OUTCOME MEASURE: All-cause mortality while hospitalised. RESULTS: The full model that included information on demographics, comorbidities, laboratory results, and vital signs had good discrimination (C-index=0.87) and was well calibrated, with some overpredictions for the most at-risk patients. Results were similar on the training and test sets, suggesting that there was little overfitting. Age was the most important risk factor. The performance of models that included all demographics and comorbidities (C-index=0.79) was only slightly better than a model that only included age (C-index=0.76). Across the study period, predicted mortality was 1.3% for patients aged 18 years old, 8.9% for 55 years old and 28.7% for 85 years old. Predicted mortality across all ages declined over the study period from 22.4% by March to 14.0% by May. CONCLUSION: Age was the most important predictor of all-cause mortality, although vital signs and laboratory results added considerable prognostic information, with oxygen saturation, temperature, respiratory rate, lactate dehydrogenase and white cell count being among the most important predictors. Demographic and comorbidity factors did not improve model performance appreciably. The full model had good discrimination and was reasonably well calibrated, suggesting that it may be useful for assessment of prognosis.

Regional variations in adverse event reporting rates and ACR responses in placebo/standard-of-care arms of rheumatoid arthritis trials.

OBJECTIVE: Clinical trials are increasingly globalized, and adverse event (AE) rates and treatment responses may differ by geographical region. This study assessed regional differences in AE reporting rates and ACR response rates (ACR20/50) in patients with RA who received placebo/standard-of-care treatment in clinical trials. METHODS: Patients from the placebo arms of 7 RA trials in the TransCelerate Biopharma Inc database were grouped into 5 geographical regions (Asia, Latin America, Russian Federation and Eastern Europe [RFEE], USA, and Western Europe). Differences in demographics, AE reporting rates and ACR response were evaluated using descriptive statistics and omnibus tests for significance; pairwise comparisons were made between regions, with false discovery rate correction for multiple comparisons. RESULTS: Among 970 patients included, week 12 AE rates were significantly lower in the RFEE than in Asia, Latin America and the USA (22% vs 51%, 49% and 53%, respectively; P < 0.05 after false discovery rate correction). Similar differences in AE rates across geographical regions were seen at week 52. Among 747 patients with ACR data, the lowest response rates were observed in the USA (ACR20, 22%) and RFEE (ACR50, 3%); the highest response rates were seen in Western Europe (ACR20, 43%) and Latin America (ACR50, 15%). Only the differences in ACR50 response between the RFEE and Latin America remained significant after false discovery rate correction. CONCLUSION: These placebo/standard-of-care arm data revealed significant regional differences in AE reporting rates and ACR50 response rates. Regional distribution of patients should be considered when conducting RA clinical trials, particularly during recruitment.

FcRn mediates fast recycling of endocytosed albumin and IgG from early macropinosomes in primary macrophages.

The neonatal Fc receptor (FcRn) rescues albumin and IgG from degradation following endocytosis and thereby extends the half-life of these plasma proteins. However, the pathways for the uptake of these soluble FcRn ligands, and the recycling itinerary of the FcRn-ligand complexes, have not been identified in primary cells. Here, we have defined the recycling of human albumin and IgG in primary mouse macrophages selectively expressing the human FcRn. Albumin is internalised by macropinocytosis; in the absence of FcRn, internalised albumin is rapidly degraded, while in the presence of FcRn albumin colocalises to SNX5-positive membrane domains and is partitioned into tubules emanating from early macropinosomes for delivery in transport carriers to the plasma membrane. Soluble monomeric IgG was also internalised by macropinocytosis and rapidly recycled by the same pathway. In contrast, the fate of IgG bound to surface Fcγ receptors differed from monomeric IgG endocytosed by macropinocytosis. Overall, our findings identify a rapid recycling pathway for FcRn ligands from early macropinosomes to the cell surface of primary cells.

Risk Associated with Cumulative Oral Glucocorticoid Use in Patients with Giant Cell Arteritis in Real-World Databases from the USA and UK.

INTRODUCTION: Treatment of giant cell arteritis (GCA) involves immediate initiation of high-dose glucocorticoid therapy with slow tapering of the dose over many months. Chronic exposure to glucocorticoids is associated with serious comorbidities. The objective of this analysis was to determine the glucocorticoid exposure and risk of glucocorticoid-related adverse events (AEs) in real-world patients with GCA. METHODS: Data from the Truven Healthcare MarketScan® database (from January 1, 2000, to June 30, 2015) and the Clinical Practice Research Datalink (CPRD; from January 1, 1995, to August 31, 2013) were used to retrospectively analyze patients aged ≥ 50 years with GCA in the USA and UK, respectively. Outcomes included oral glucocorticoid use (cumulative prednisone-equivalent exposure), glucocorticoid-related AEs and the association of AE risk with glucocorticoid exposure over 52 weeks. RESULTS: Of the 4804 patients in the US MarketScan database and 3973 patients in the UK CPRD database included, 71.3 and 74.6% were women and mean age was 73.4 and 73.0 years, respectively. Median starting glucocorticoid dose and cumulative glucocorticoid dose at 52 weeks were 20-50 mg/day and 4000-4800 mg, respectively. The most frequent glucocorticoid-related AEs were hypertension and eye, bone health, and glucose tolerance conditions. In the first year after diagnosis, the likelihood of any glucocorticoid-related AE was significantly increased for each 1 g increase in cumulative glucocorticoid dose in the US and UK cohorts (odds ratio [95% CI], 1.170 [1.063, 1.287] and 1.06 [1.03, 1.09], respectively; P < 0.05 for both). Similar trends were observed for the risk of glucocorticoid-related AEs over full follow-up (mean, USA: 3.9 years, UK: 6.3 years). CONCLUSIONS: In real-world patients with GCA, increased cumulative glucocorticoid exposure was associated with an increased risk of glucocorticoid-related AEs. FUNDING: F. Hoffmann-La Roche Ltd. Plain language summary available for this article.

rIgG1 Fc Hexamer Inhibits Antibody-Mediated Autoimmune Disease via Effects on Complement and FcγRs.

Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-µTP-L309C) generated by fusion of the IgM µ-tailpiece to the C terminus of human IgG1 Fc. Fc-µTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-µTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-µTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-µTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-µTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.

Half-life-extended recombinant coagulation factor IX-albumin fusion protein is recycled via the FcRn-mediated pathway.

The neonatal Fc receptor (FcRn) has a pivotal role in albumin and IgG homeostasis. Internalized IgG captured by FcRn under acidic endosomal conditions is recycled to the cell surface where exocytosis and a shift to neutral pH promote extracellular IgG release. Although a similar mechanism is proposed for FcRn-mediated albumin intracellular trafficking and recycling, this pathway is less well defined but is relevant to the development of therapeutics exploiting FcRn to extend the half-life of short-lived plasma proteins. Recently, a long-acting recombinant coagulation factor IX-albumin fusion protein (rIX-FP) has been approved for the management of hemophilia B. Fusion to albumin potentially enables internalized proteins to engage FcRn and escape lysosomal degradation. In this study, we present for the first time a detailed investigation of the FcRn-mediated recycling of albumin and the albumin fusion protein rIX-FP. We demonstrate that following internalization via FcRn at low pH, rIX-FP, like albumin, is detectable within the early endosome and rapidly (within 10-15 min) traffics into the Rab11+ recycling endosomes, from where it is exported from the cell. Similarly, rIX-FP and albumin taken up by fluid-phase endocytosis at physiological pH traffics into the Rab11+ recycling compartment in FcRn-positive cells but into the lysosomal compartment in FcRn-negative cells. As expected, recombinant factor IX (without albumin fusion) and an FcRn interaction-defective albumin variant localized to the lysosomal compartments of both FcRn-expressing and nonexpressing cells. These results indicate that FcRn-mediated recycling via the albumin moiety is a mechanism for the half-life extension of rIX-FP observed in clinical studies.

Persistence and switching patterns of oral migraine prophylactic medications among patients with chronic migraine: A retrospective claims analysis.

Background Migraine prevention guidelines recommend oral prophylactic medications for patients with frequent headache. This study examined oral migraine preventive medication (OMPM) treatment patterns by evaluating medication persistence, switching, and re-initiation in patients with chronic migraine (CM). Methods A retrospective US claims analysis (Truven Health MarketScan® Databases) evaluated patients ≥18 years old diagnosed with CM who had initiated an OMPM between 1 January, 2008 and 30 September, 2012. Treatment persistence was measured at six and 12 months' follow-up. Time-to-discontinuation was assessed for each OMPM and compared using Cox regression models. Among those who discontinued, the proportion that switched OMPMs within 60 days or re-initiated treatment between 61 to 365 days, and their associated persistence rates, were also assessed. Results A total of 8707 patients met the inclusion/exclusion criteria. Persistence to the initial OMPM was 25% at six months and 14% at 12 months. Based on Kaplan-Meier curves, a sharp decline of patients discontinuing was observed by 30 days, and approximately half discontinued by 60 days. Similar trends in time-to-discontinuation were seen following the second or third OMPM. Amitriptyline, gabapentin, and nortriptyline had significantly higher likelihood of non-persistence compared with topiramate. Among patients who discontinued, 23% switched to another prophylactic and 41% re-initiated therapy within one year. Among patients who switched, persistence was between 10 to 13% and among re-initiated patients, persistence was between 4 to 8% at 12 months. Conclusions Persistence to OMPMs is poor at six months and declines further by 12 months. Switching between OMPMs is common, but results indicate that persistence worsens as patients cycle through various OMPMs.

Health care resource utilization following initiation of a triptan: a retrospective claims analysis.

BACKGROUND: 23 million Americans suffer from migraine headaches, incurring more than $1 billion in direct medical costs each year (with another $13 billion in indirect productivity losses). Triptans are the most common treatment of choice for these patients; however, adherence and persistence to triptans are poor. Partly due to poor adherence to therapy, the ability of triptans to reduce the utilization of other medical services and prescription drugs remains unclear. OBJECTIVES: To (a) assess changes in the utilization of medical services and relevant prescription drugs after patients suffering from episodic migraines begin triptan therapy and (b) further investigate the relationship between concomitant opioid use among triptan-treated migraine patients and further utilization of medical services and prescription drugs. METHODS: A retrospective analysis of pharmacy and medical insurance claims was carried out using a large and nationally representative database. The utilization patterns of episodic migraine patients were observed for 12 to 24 months prior to their first triptan prescription and 12 to 24 months following that prescription. Resource utilization included physician office visits, diagnostic imaging, emergency room use, inpatient hospitalization, opioid prescriptions, migraine prophylaxis prescriptions, and acetaminophen or nonsteroidal anti-inflammatory prescriptions. Results were stratified according to triptan-switching behavior. RESULTS: The analytic sample included 9,521 migraine patients who were followed for a median of 550 days before and after their first triptan fill. 40.9% of these patients filled their triptan prescriptions only once (index fill). Another 40.3% filled a triptan prescription at least twice and never switched their triptan brand. 15.6% of patients switched their triptan prescriptions once, and 3.2% of patients switched their triptan prescriptions twice or more. The only group to display significant reductions in resource utilization following the prescription of a triptan was the cohort that never refilled the medication, potentially suggestive of misdiagnosis. Either no significant change or a significant increase in resource utilization was seen in all other cases. The ability of triptans to reduce resource utilization seemed to be lower among patients who switched triptans more often. Patients that concomitantly used opioid medications in addition to triptans also used significantly more resources than migraine patients who were not treated with opioids. CONCLUSION: Contrary to the findings of some previous research, the initiation of triptan therapy did not significantly reduce the utilization of migraine-related medical services or other relevant prescription drugs in this retrospective claims analysis. This may have been due to higher and more realistic rates of triptan switching and discontinuation. Consistent with previous findings, patients using concomitant opioids used more migraine-related health care resources.

Health claims database study of cyclosporine ophthalmic emulsion treatment patterns in dry eye patients.

BACKGROUND: Dry eye is a multifactorial, symptomatic disease associated with ocular surface inflammation and tear film hyperosmolarity. This study was designed to assess patterns of topical cyclosporine ophthalmic emulsion 0.05% (Restasis®) use in dry eye patients and determine if there were any differences in use based on whether dry eye is physician-coded as a primary or nonprimary diagnosis. METHODS: Records for adult patients with a diagnosis of dry eye at an outpatient visit from January 1, 2008 to December 31, 2009 were selected from Truven Health MarketScan® Research Databases. The primary endpoint was percentage of patients with at least one primary versus no primary dry eye diagnosis who filled a topical cyclosporine prescription. Data analyzed included utilization of topical corticosteroids, oral tetracyclines, and punctal plugs. RESULTS: The analysis included 576,416 patients, accounting for 875,692 dry eye outpatient visits: 74.7% were female, 64.2% were ages 40-69 years, and 84.4% had at least one primary dry eye diagnosis. During 2008-2009, 15.9% of dry eye patients with a primary diagnosis versus 6.5% with no primary diagnosis filled at least one cyclosporine prescription. For patients who filled at least one prescription, the mean months' supply of cyclosporine filled over 12 months was 4.44. Overall, 33.9% of dry eye patients filled a prescription for topical cyclosporine, topical corticosteroid, or oral tetracycline over 2 years. CONCLUSION: Patients with a primary dry eye diagnosis were more likely to fill a topical cyclosporine prescription. Although inflammation is key to the pathophysiology of dry eye, most patients seeing a physician for dry eye may not receive anti-inflammatory therapies.

Human perforin mutations and susceptibility to multiple primary cancers.

Loss-of-function mutations in the gene coding for perforin (PRF1) markedly reduce the ability of cytotoxic T lymphocytes and natural killer cells to kill target cells, causing immunosuppression and impairing immune regulation. In humans, nearly half of the cases of type 2 familial hemophagocytic lymphohistiocytosis are due to bi-allelic PRF1 mutations. The partial inactivation of PRF1 due to mutations that promote protein misfolding or the common hypomorphic allele coding for the A91V substitution have been associated with lymphoid malignancies in childhood and adolescence. To investigate whether PRF1 mutations also predispose adults to cancer, we genotyped 566 individuals diagnosed with melanoma (101), lymphoma (65), colorectal carcinoma (30) or ovarian cancer (370). The frequency of PRF1 genotypes was similar in all disease groups and 424 matched controls, indicating that the PRF1 status is not associated with an increased susceptibility to these malignancies. However, four out of 15 additional individuals diagnosed with melanoma and B-cell lymphoma during their lifetime expressed either PRF1A91V or the rare pathogenic PRF1R28C variant (p = 0.04), and developed melanoma relatively early in life. Both PRF1A91V- and PRF1R28C-expressing lymphocytes exhibited severely impaired but measurable cytotoxic function. Our results suggest that defects in human PRF1 predispose individuals to develop both melanoma and lymphoma. However, these findings require validation in larger patient cohorts.

Fatal immune dysregulation due to a gain of glycosylation mutation in lymphocyte perforin.

Mutations in the perforin gene (PRF1) are a common cause of the fatal immune dysregulation disorder, familial hemophagocytic lymphohistiocytosis (type 2 FHL, FHL2). Here we report a female infant born with biallelic PRF1 mutations: a novel substitution, D49N, and a previously identified in-frame deletion, K285del. We assessed the effects of each mutation on the cytotoxicity of human NK cells in which the expression of endogenous perforin was ablated with miR30-based short hairpin (sh) RNAs. Both mutations were detrimental for function, thereby explaining the clinically severe presentation and rapidly fatal outcome. We demonstrate that D49N exerts its deleterious effect by generating an additional (third) N-linked glycosylation site, resulting in protein misfolding and degradation in the killer cell. Our data provide a rationale for treating some cases of type 2 familial hemophagocytic lymphohistiocytosis, based on the pharmacologic inhibition or modification of glycosylation.

Protection from endogenous perforin: glycans and the C terminus regulate exocytic trafficking in cytotoxic lymphocytes.

Cytotoxic lymphocyte-mediated apoptosis is dependent on the delivery of perforin to secretory granules and its ability to form calcium-dependent pores in the target cell after granule exocytosis. It is unclear how cytotoxic lymphocytes synthesize and store perforin without incurring damage or death. We discovered that the extreme C terminus of perforin was essential for rapid trafficking from the endoplasmic reticulum to the Golgi compartment. Substitution of the C-terminal tryptophan residue resulted in retention of perforin in the ER followed by calcium-dependent toxic activity that eliminated host cells. We also found that N-linked glycosylation of perforin was critical for transport from the Golgi to secretory granules. Overall, an intact C terminus and N-linked glycosylation provide accurate and efficient export of perforin from the endoplasmic reticulum to the secretory granules and are critical for cytotoxic lymphocyte survival.

Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer.

The pore-forming protein perforin is critical for defense against many human pathogens and for preventing a catastrophic collapse of immune homeostasis, manifested in infancy as Type 2 familial hemophagocytic lymphohistiocytosis (FHL). However, no evidence has yet linked defective perforin cytotoxicity with cancer susceptibility in humans. Here, we examined perforin function in every patient reported in the literature who lived to at least 10 years of age without developing FHL despite inheriting mutations in both of their perforin (PRF1) alleles. Our analysis showed that almost 50% of these patients developed at least 1 hematological malignancy in childhood or adolescence. The broad range of pathologies argued strongly against a common environmental or viral cause for the extraordinary cancer incidence. Functionally, what distinguished these patients was their inheritance of PRF1 alleles encoding temperature-sensitive missense mutations. By contrast, truly null missense mutations with no rescue at the permissive temperature were associated with the more common severe presentation with FHL in early infancy. Our study provides the first mechanistic evidence for a link between defective perforin-mediated cytotoxicity and cancer susceptibility in humans and establishes the paradigm that temperature sensitivity of perforin function is a predictor of FHL severity.

The molecular basis for perforin oligomerization and transmembrane pore assembly.

Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into perforin's mechanism of action.

Health coverage of low-income citizen and noncitizen wage earners: sources and disparities.

The health coverage of low-income workers represents an area of continuing disparities in the United States system of health insurance. Using the 2001 California Health Interview Survey, we estimate the effect of low-income wage earners' citizenship and gender on the odds of obtaining primary employment-based health insurance (EBHI), dependent EBHI, public program coverage, and coverage from any source. We find that noncitizen men and women who comprise 40% of California's low-income workforce, share the disadvantage of much lower rates of insurance coverage, compared to naturalized and U.S.-born citizens. However, poor coverage rates of noncitizen men, regardless of permanent residency status, result from the cumulative disadvantage in obtaining dependent EBHI and public insurance. If public policies designed to provide a health care safety net fail to address the health care coverage needs of low-wage noncitizens, health disparities will continue to increase in this group that contributes essentially to the U.S. economy.

Evidence for a role of tumor-derived laminin-511 in the metastatic progression of breast cancer.

Most studies investigating laminins (LMs) in breast cancer have focused on LM-111 or LM-332. Little is known, however, about the expression and function of alpha5 chain-containing LM-511/521 during metastatic progression. Expression of LM-511/521 subunits was examined in genetically related breast tumor lines and corresponding primary tumors and metastases in a syngeneic mouse model using real-time quantitative polymerase chain reaction, in situ hybridization, and immunohistochemistry. The results from our investigation indicate that LM-511 rather than LM-111, -332, or -521 correlates with metastatic potential in mouse mammary tumors. LM-511 was a potent adhesive substrate for both murine and human breast carcinoma cells and promoted strong haptotactic responses in metastatic lines. Haptotaxis was mediated by alpha3 integrin in both MCF-7 and MDA-MB-231 cells and was strongly inhibited by blocking antibodies against this integrin subunit. However, whereas nonmetastatic MCF-7 cells migrated toward LM-511 primarily via alpha3beta1 integrin, results from antibody perturbation experiments and flow cytometry analysis suggest that this response is mediated by an as yet unidentified alpha3beta integrin heterodimer (other than alpha3beta1) in MDA-MB-231 cells. These results are consistent with earlier reports implicating alpha3 integrins in breast cancer progression and support the role of LM-511 as a functional substrate regulating breast cancer metastasis.

Perforin activity and immune homeostasis: the common A91V polymorphism in perforin results in both presynaptic and postsynaptic defects in function.

Perforin (PRF), a pore-forming protein expressed in cytotoxic lymphocytes, plays a key role in immune surveillance and immune homeostasis. The A91V substitution has a prevalence of 8% to 9% in population studies. While this variant has been suspected of predisposing to various disorders of immune homeostasis, its effect on perforin's function has not been elucidated. Here we complemented, for the first time, the cytotoxic function of perforin-deficient primary cytotoxic T lymphocytes (CTLs) with wild-type (hPRF-WT) and A91V mutant (hPRF-A91V) perforin. The cytotoxicity of hPRF-A91V-expressing cells was about half that of hPRF-WT-expressing counterparts and coincided with a moderate reduction in hPRF-A91V expression. By contrast, the reduction in cytotoxic function was far more pronounced (more than 10-fold) when purified proteins were tested directly on target cells. The A91V substitution can therefore be manifested by abnormalities at both the lymphocyte (presynaptic) and target cell (postsynaptic) levels. However, the severe intrinsic defect in activity can be partly rescued by expression in the physiological setting of an intact CTL. These findings provide the first direct evidence that hPRF-A91V is functionally abnormal and provides a rationale for why it may be responsible for disordered immune homeostasis if inherited with another dysfunctional perforin allele.