BIOSIMILARS AND NEW INSULIN VERSIONS.

OBJECTIVE: To provide clinicians with an overview of similar biologic products including biosimilars and new insulin versions available in the U.S. and of key issues associated with such products, including differences in manufacturing and regulatory approaches and their impact on clinical use. METHODS: We reviewed the relevant clinical and regulatory literature. RESULTS: Patent protections for many biologics including several insulin preparations have or will expire shortly. This opens the door for new insulin versions to enter the U.S. and global marketplace. The development, manufacturing, and approval process for similar biologic products is more complex than for generic versions of small molecules. Most similar biologic products in the U.S. will be submitted for approval under section 351(k), a newly created biosimilar regulatory pathway. However, some biologics, including new insulin versions, will be submitted via the existing 505(b)(2) regulatory pathway. These regulatory pathways have implications for how such products may be labeled, how they may be dispensed, and how patients may perceive them. The immunogenicity of biologics can affect safety and efficacy and can be altered through subtle changes in manufacturing. With the arrival of new insulin versions, health care providers will need to understand the implications of interchangeability, therapeutic equivalence, substitution, switching, and new delivery devices. CONCLUSION: An understanding of the above topics will be important as physicians, payers, and patients choose between similar versions of a reference listed biologic product.

Severely blunted allergen-induced pulmonary Th2 cell response and lung hyperresponsiveness in type 1 transient receptor potential channel-deficient mice.

Transient receptor potential channels (TRPCs) are widely expressed and regulate Ca²âº entry in the cells that participate in the pathophysiology of airway hyperreactivity, inflammation, and remodeling. In vitro studies point to a role for TRPC1-mediated Ca²âº signaling in several of these cell types; however, physiological evidence is lacking. Here we identify TRPC1 signaling as proinflammatory and a regulator of lung hyperresponsiveness during allergen-induced pulmonary response. TRPC1-deficient (Trpc1(-/-)) mice are hyposensitive to methacholine challenge and have significantly reduced allergen-induced pulmonary leukocyte infiltration coupled with an attenuated T helper type 2 (Th2) cell response. Upon in vitro allergen exposure, Trpc1(-/-) splenocytes show impaired proliferation and T cell receptor-induced IL-2 production. A high number of germinal centers in spleens of Trpc1(-/-) mice and elevated levels of immunoglobulins in their serum are indicative of dysregulated B cell function and homeostasis. Thus we propose that TRPC1 signaling is necessary in lymphocyte biology and in regulation of allergen-induced lung hyperresponsiveness, making TRPC1 a potential target for treatment of immune diseases and asthma.

Modulation of allergic airway inflammation by the oral pathogen Porphyromonas gingivalis.

Accumulating evidence suggests that bacteria associated with periodontal disease may exert systemic immunomodulatory effects. Although the improvement in oral hygiene practices in recent decades correlates with the increased incidence of asthma in developed nations, it is not known whether diseases of the respiratory system might be influenced by the presence of oral pathogens. The present study sought to determine whether subcutaneous infection with the anaerobic oral pathogen Porphyromonas gingivalis exerts a regulatory effect on allergic airway inflammation. BALB/c mice sensitized and subsequently challenged with ovalbumin exhibited airway hyperresponsiveness to methacholine aerosol and increased airway inflammatory cell influx and Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13) content relative to those in nonallergic controls. Airway inflammatory cell and cytokine contents were significantly reduced by establishment of a subcutaneous infection with P. gingivalis prior to allergen sensitization, whereas serum levels of ovalbumin-specific IgE and airway responsiveness were not altered. Conversely, subcutaneous infection initiated after allergen sensitization did not alter inflammatory end points but did reduce airway responsiveness in spite of increased serum IgE levels. These data provide the first direct evidence of a regulatory effect of an oral pathogen on allergic airway inflammation and responsiveness. Furthermore, a temporal importance of the establishment of infection relative to allergen sensitization is demonstrated for allergic outcomes.

Ghostwriting in biomedicine: a review of the published literature.

Introduction: The systematic review of biomedical ghostwriting has proven challenging due to problems in consistency and in study design. Moreover, authorship guidelines established by the International Committee of Medical Journal Editors (ICMJE) may have inadvertently created opportunities to potentiate ghostwriting. Given continued interest in ghostwriting by the International Society of Medical Publication Professionals (ISMPP) and other organizations, we undertook an analysis of ghostwriting in the biomedical literature.Methods: We searched PubMed (search terms: ghost writ*, ghostwrit*, ghost writer, ghostwriter, ghostwriting and ghost writing). Results, including abstracts, were reviewed for relevance (relationship to ghostwriting in biomedical journals) to aid in removal of inapplicable work and duplicate publications. After review, we consolidated expert opinions for publication professionals.Results: Overlap was poor across search terms; of 181 unique papers identified, most (112/181) were opinion pieces. An increasing number of papers are using the term "ghostwriting" to describe genetics as well as diverse phenomena of misattributed authorship, including "ghost authorship". Eight primary studies and 1 systematic review of ghostwriting incidence were identified, reporting prevalence ranging from <1% to 91%, in varied settings using differing methods and definitions of ghostwriting. Suggestions for avoiding ghostwriting include early consensus building and better definitions of authorship among manuscript teams.Discussion: The prevalence and definition of ghostwriting remain unclear. Increased transparency and auditable authorship practices that align with specific guidelines may aid in the avoidance of ghostwriting. In addition, MeSH or clearer indexing terms may be helpful to separate usages of ghostwriting in scientific settings (e.g. genetic research) versus biomedical publishing.

Male sex hormones exacerbate lung function impairment after bleomycin-induced pulmonary fibrosis.

The roles of sex hormones as modulators of lung function and disease have received significant attention as differential sex responses to various lung insults have been recently reported. The present study used a bleomycin-induced pulmonary fibrosis model in C57BL/6 mice to examine potential sex differences in physiological and pathological outcomes. Endpoints measured included invasive lung function assessment, immunological response, lung collagen deposition, and a quantitative histological analysis of pulmonary fibrosis. Male mice had significantly higher basal static lung compliance than female mice (P < 0.05) and a more pronounced decline in static compliance after bleomycin administration when expressed as overall change or percentage of baseline change (P < 0.05). In contrast, there were no significant differences between the sexes in immune cell infiltration into the lung or in total lung collagen content after bleomycin. Total lung histopathology scores measured using the Ashcroft method did not differ between the sexes, while a quantitative histopathology scoring system designed to determine where within the lung the fibrosis occurred indicated a tendency toward more fibrosis immediately adjacent to airways in bleomycin-treated male versus female mice. Furthermore, castrated male mice exhibited a female-like response to bleomycin while female mice given exogenous androgen exhibited a male-like response. These data indicate that androgens play an exacerbating role in decreased lung function after bleomycin administration, and traditional measures of fibrosis may miss critical differences in lung function between the sexes. Sex differences should be carefully considered when designing and interpreting experimental models of pulmonary fibrosis in mice.

Role of soluble epoxide hydrolase in postischemic recovery of heart contractile function.

Cytochrome P450 epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (Ephx2, sEH). To examine the functional role of sEH in the heart, mice with targeted disruption of the Ephx2 gene were studied. Hearts from sEH null mice have undetectable levels of sEH mRNA and protein and cannot convert EETs to DHETs. sEH null mice have normal heart anatomy and basal contractile function, but have higher fatty acid epoxide:diol ratios in plasma and cardiomyocyte cell culture media compared with wild type (WT). sEH null hearts have improved recovery of left ventricular developed pressure (LVDP) and less infarction compared with WT hearts after 20 minutes ischemia. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 to 100 nmol/L) before ischemia abolishes this cardioprotective phenotype. Inhibitor studies demonstrate that perfusion with phosphatidylinositol-3 kinase (PI3K) inhibitors wortmannin (200 nmol/L) or LY294002 (5 micromol/L), the ATP-sensitive K+ channel (K(ATP)) inhibitor glibenclamide (1 micromol/L), the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (100 to 200 micromol/L), or the Ca2+-sensitive K+ channel (K(Ca)) inhibitor paxilline (10 micromol/L) abolishes the cardioprotection in sEH null hearts. Consistent with increased activation of the PI3K cascade, sEH null mice exhibit increased cardiac expression of glycogen synthase kinase-3beta (GSK-3beta) phospho-protein after ischemia. Together, these data suggest that targeted disruption of sEH increases the availability of cardioprotective EETs that work by activating PI3K signaling pathways and K+ channels.

It's all about sex: gender, lung development and lung disease.

Accumulating evidence suggests that gender affects the incidence, susceptibility and severity of several lung diseases. Gender also influences lung development and physiology. Data from both human and animal studies indicate that sex hormones might contribute to disease pathogenesis or serve as protective factors, depending on the disease involved. In this review, the influence of gender and sex hormones on lung development and pathology will be discussed, with specific emphasis on pulmonary fibrosis, asthma and cancer.

Randomized Clinical Trial Comparing Basal Insulin Peglispro and Insulin Glargine in Patients With Type 2 Diabetes Previously Treated With Basal Insulin: IMAGINE 5.

OBJECTIVE: To evaluate the efficacy and safety of basal insulin peglispro (BIL) versus insulin glargine in patients with type 2 diabetes (hemoglobin A1c [HbA1c] ≤9% [75 mmol/mol]) treated with basal insulin alone or with three or fewer oral antihyperglycemic medications. RESEARCH DESIGN AND METHODS: This 52-week, open-label, treat-to-target study randomized patients (mean HbA1c 7.42% [57.6 mmol/mol]) to BIL (n = 307) or glargine (n = 159). The primary end point was change from baseline HbA1c to 26 weeks (0.4% [4.4 mmol/mol] noninferiority margin). RESULTS: At 26 weeks, reduction in HbA1c was superior with BIL versus glargine (-0.82% [-8.9 mmol/mol] vs. -0.29% [-3.2 mmol/mol]; least squares mean difference -0.52%, 95% CI -0.67 to -0.38 [-5.7 mmol/mol, 95% CI -7.3 to -4.2; P < 0.001); greater reduction in HbA1c with BIL was maintained at 52 weeks. More BIL patients achieved HbA1c <7% (53 mmol/mol) at weeks 26 and 52 (P < 0.001). With BIL versus glargine, nocturnal hypoglycemia rate was 60% lower, more patients achieved HbA1c <7% (53 mmol/mol) without nocturnal hypoglycemia at 26 and 52 weeks (P < 0.001), and total hypoglycemia rates were lower at 52 weeks (P = 0.03). At weeks 26 and 52, glucose variability was lower (P < 0.01), basal insulin dose was higher (P < 0.001), and triglycerides and aminotransferases were higher with BIL versus glargine (P < 0.05). Liver fat content (LFC), assessed in a subset of patients (n = 162), increased from baseline with BIL versus glargine (P < 0.001), with stable levels between 26 and 52 weeks. CONCLUSIONS: BIL provided superior glycemic control versus glargine, with reduced nocturnal and total hypoglycemia, lower glucose variability, and increased triglycerides, aminotransferases, and LFC.

Tipping the balance towards tolerance: the basis for therapeutic immune modulation by gold?

Gold salts have long been used in the treatment of rheumatoid arthritis. However, the basis for their therapeutic immune-modulating properties has never been satisfactorily explained. Furthermore, treatments are often marred by the development of adverse immune reactions such as hypersensitivity and even exacerbation of autoimmunity. We would like to propose a hypothesis to explain the basis for both the beneficial and adverse immune-modulating effects of gold in the treatment of rheumatoid arthritis. If accepted, this hypothesis will allow for the development of safer and more effective treatments with gold salts. The principle underlying this hypothesis also has broader implications for how immune hypersensitivity and tolerance are perceived.

Immunochemical detection of flucloxacillin adduct formation in livers of treated rats.

Flucloxacillin is a semi-synthetic penicillin widely used in the prophylaxis and treatment of staphylococcal infections. Severe liver reactions, characterised by delayed cholestatic hepatitis and a prolonged course of recovery, are associated with flucloxacillin therapy. Clinical findings are suggestive of an immune mediated reaction but there exists little supporting experimental evidence. The formation of drug modified hepatic protein adducts has been proposed to play an important role in the hepatotoxicity of many drugs. The aim of the present study was to investigate whether flucloxacillin treatment results in adduct formation in vivo. Flucloxacillin was conjugated to rabbit serum albumin by formation of a penicilloyl determinant and used as an immunogen to raise a polyclonal antiserum specific for flucloxacillin-modified proteins. Antibody specificity was confirmed by competitive enzyme-linked immunosorbent assay (ELISA) with free drug. The antiserum was used in combination with western blotting to detect adduct formation in the livers of flucloxacillin treated rats. Western blot analysis of rat liver subcellular fractions revealed the formation of six flucloxacillin adducts in various subcellular fractions. These studies demonstrate for the first time that treatment with flucloxacillin results in the formation of hepatic protein adducts.

Cyclooxygenase enzymes in allergic inflammation and asthma.

The cyclooxygenase enzyme system produces eicosanoids which mediate many important physiological and pathological functions. Experimental and clinical data suggest a role for this enzyme system in the pathogenesis of allergic inflammation and asthma. This article focuses on the function of this pathway in the lung, reviews evidence implicating the involvement of this pathway in asthma and allergic airway inflammation, and discusses implications for the treatment of asthmatics with cyclooxygenase inhibitors.

A randomized, cross-over comparison of preference between two reusable insulin pen devices in pen-naïve adults with diabetes.

OBJECTIVE: To evaluate final preference and ease-of-use attributes of two reusable pen injectors, HPS (HumaPen Savvio) and HPL (HumaPen Luxura), in adults with type 1 or type 2 diabetes. RESEARCH DESIGN AND METHODS: This was a 1 day, randomized, two-period crossover, open-label, simulated-injection study in 203 pen-naïve subjects (mean age 58.4 years). MAIN OUTCOME MEASURES: Functional and ease-of-use attributes of insulin pen injectors were evaluated using a 16-item survey (7 point scale) where higher scores reflected greater preference and equal scores reflected no preference. The primary objective was final pen preference, with statistical gate-keeping to the ease of detecting an insufficient remaining dose (IRD) of insulin upon dose selection. RESULTS: For final overall pen preference, HPS was chosen by 150 of 203 subjects (73.9%, 95% confidence interval [CI] = 67.3%-79.8%). For the IRD item, 'It is easy to know when there is not enough insulin left in the cartridge for the dose I need before I inject', HPS was preferred by 94 of 107 subjects with a preference (87.9%, 95% CI = 80.1%-93.4%). In 14 of the remaining 15 survey items, 64.3% to 87.7% of subjects with a preference statistically significantly preferred HPS over HPL. To confirm the results, subjects with no preference for either pen, which ranged between 95 and 148, were included in a Bayesian analysis. KEY LIMITATIONS: Injection simulation, use of an unvalidated survey, and office setting which did not allow for direct clinical experience with the devices. CONCLUSIONS: The majority of pen-naïve subjects preferred HPS over HPL. For all ease-of-use attributes, the majority of subjects with a preference chose HPS over HPL. Some attributes of both pens were equally acceptable, as many subjects had no preference.

The effect of adjusting for baseline hypoglycemia when analyzing hypoglycemia data: a systematic analysis of 15 diabetes clinical trials.

BACKGROUND: Baseline hypoglycemia rates are generally not collected or included as a covariate in statistical models used for analyzing hypoglycemia data. The objective of the present study was to examine the effect of adjusting for baseline hypoglycemia on estimation efficiency and statistical power. SUBJECTS AND METHODS: A post hoc analysis of data from 15 insulin trials, including patients with type 1 diabetes mellitus (T1DM) (n=210), previously insulin-treated type 2 diabetes mellitus (T2DM) (n=1,511), or T2DM and previously insulin-naive (n=1,075). Hypoglycemic episodes were analyzed with a negative binomial regression model. RESULTS: Baseline nocturnal hypoglycemia rate was significantly correlated with post-baseline nocturnal hypoglycemia rate in previously insulin-treated patients with T1DM and T2DM (correlation range, 0.37-0.63; P<0.001). Adjusting for baseline hypoglycemia resulted in a reduction in the SE for negative binomial regression for previously insulin-treated patients with T1DM and T2DM (range, 2.2-11.8%) and increased statistical power. Modeling of lengthening the lead-in period increases the correlation between baseline and post-baseline hypoglycemia event rate and statistical power. CONCLUSIONS: Baseline hypoglycemia rate is significantly correlated with post-baseline hypoglycemia rate for patients with diabetes treated with insulin prior to randomization. The length of the lead-in period can impact correlations between baseline and post-baseline data, and adjustment for baseline hypoglycemia may improve the estimation efficiency for hypoglycemia data analyses in clinical trials.

Pharmacologic inhibition of COX-1 and COX-2 in influenza A viral infection in mice.

BACKGROUND: We previously demonstrated that cyclooxygenase (COX)-1 deficiency results in greater morbidity and inflammation, whereas COX-2 deficiency leads to reduced morbidity, inflammation and mortality in influenza infected mice. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of COX-1 and COX-2 inhibitors in influenza A viral infection. Mice were given a COX-1 inhibitor (SC-560), a COX-2 inhibitor (celecoxib) or no inhibitor beginning 2 weeks prior to influenza A viral infection (200 PFU) and throughout the course of the experiment. Body weight and temperature were measured daily as indicators of morbidity. Animals were sacrificed on days 1 and 4 post-infection and bronchoalveolar lavage (BAL) fluid was collected or daily mortality was recorded up to 2 weeks post-infection. Treatment with SC-560 significantly increased mortality and was associated with profound hypothermia and greater weight loss compared to celecoxib or control groups. On day 4 of infection, BAL fluid cells were modestly elevated in celecoxib treated mice compared to SC-560 or control groups. Viral titres were similar between treatment groups. Levels of TNF-alpha and G-CSF were significantly attenuated in the SC-560 and celecoxib groups versus control and IL-6 levels were significantly lower in BAL fluid of celecoxib treated mice versus control and versus the SC-560 group. The chemokine KC was significantly lower in SC-560 group versus control. CONCLUSIONS/SIGNIFICANCE: Treatment with a COX-1 inhibitor during influenza A viral infection is detrimental to the host whereas inhibition of COX-2 does not significantly modulate disease severity. COX-1 plays a critical role in controlling the thermoregulatory response to influenza A viral infection in mice.

The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies.

Numerous animal studies have revealed significant effects of sex and sex hormones on normal lung development, lung physiology, and various lung diseases. The primary goal of this review is to summarize knowledge to date on the effects of sex and sex hormones on lung development, physiology, and disease in animals. Specific emphasis will be placed on fibrosis, allergic airway disease, acute lung injury models, respiratory infection, and lung toxicology studies.

Cyclooxygenase-2 deficiency exacerbates bleomycin-induced lung dysfunction but not fibrosis.

Cyclooxygenase (COX)-derived eicosanoids have been implicated in the pathogenesis of pulmonary fibrosis. Uncertainty regarding the influence of COX-2 on experimental pulmonary fibrosis prompted us to clarify the fibrotic and functional effects of intratracheal bleomycin administration in mice genetically deficient in COX-2. Further, the effects of airway-specific COX-1 overexpression on fibrotic and functional outcomes in wild-type and COX-2 knockout mice were assessed. Equivalent increases in airway cell influx, lung collagen content, and histopathologic evidence of fibrosis were observed in wild-type and COX-2 knockout mice 21 d after bleomycin treatment, suggesting that COX-2 deficiency did not alter the extent or severity of fibrosis in this model. However, bleomycin-induced alterations in respiratory mechanics were more severe in COX-2 knockout mice than in wild-type mice, as illustrated by a greater decrease in static compliance compared with genotype-matched, saline-treated control mice (26 +/- 3% versus 11 +/- 4% decreases for COX-2 knockout and wild-type mice, respectively; P < 0.05). The influence of COX-1 overexpression in airway Clara cells was also examined. Whereas the fibrotic effects of bleomycin were not altered in wild-type or COX-2 knockout mice overexpressing COX-1, the exaggerated lung function decrement in bleomycin-treated COX-2 knockout mice was prevented by COX-1 overexpression and coincided with decreased airway cysteinyl leukotriene levels. Collectively, these data suggest an important regulatory role for COX-2 in the maintenance of lung function in the setting of lung fibrosis, but not in the progression of the fibrotic process per se.

Male sex hormones promote vagally mediated reflex airway responsiveness to cholinergic stimulation.

A sex disparity in airway responsiveness to cholinergic stimulation has been observed in laboratory mice in that males are considerably more responsive than females, but the basis for this difference is unclear. In this report, we demonstrate that male sex hormones promote murine airway responsiveness to cholinergic stimulation via vagus nerve-mediated reflex mechanisms. In tissue bath preparations, no sex-based differences were observed in the contractile responses of isolated tracheal and bronchial ring segments to carbachol, indicating that the mechanism(s) responsible for the in vivo sex difference is (are) absent ex vivo. Bilateral cervical vagotomy was found to abolish in vivo airway responsiveness to methacholine in male mice, whereas it did not alter the responses of females, suggesting a regulatory role for male sex hormones in promoting reflex airway constriction. To test this possibility, we next studied mice with altered circulating male sex hormone levels. Castrated male mice displayed airway responsiveness equivalent to that observed in intact females, whereas administration of exogenous testosterone to castrated males restored responsiveness, albeit not to the level observed in intact males. Administration of exogenous testosterone to intact female mice similarly enhanced responsiveness. Importantly, the promotive effects of exogenous testosterone in castrated male and intact female mice were absent when bilateral vagotomy was performed. Together, these data indicate that male sex hormones promote cholinergic airway responsiveness via a vagally mediated reflex mechanism that may be important in the regulation of airway tone in the normal and diseased lung.

Spontaneous airway hyperresponsiveness in estrogen receptor-alpha-deficient mice.

RATIONALE: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans. OBJECTIVES: To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor-deficient mice. METHODS: Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure. MEASUREMENTS AND MAIN RESULTS: Estrogen receptor-alpha knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-alpha also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge. CONCLUSIONS: These data suggest that estrogen receptor-alpha is a critical regulator of airway hyperresponsiveness in mice.

Gender differences in murine airway responsiveness and lipopolysaccharide-induced inflammation.

The roles of gender and sex hormones in lung function and disease are complex and not completely understood. The present study examined the influence of gender on lung function and respiratory mechanics in naive mice and on acute airway inflammation and hyperresponsiveness induced by intratracheal LPS administration. Basal lung function characteristics did not differ between naive males and females, but males demonstrated significantly greater airway responsiveness than females following aerosolized methacholine challenge as evidenced by increased respiratory system resistance and elastance (p < 0.05). Following LPS administration, males developed more severe hypothermia and greater airway hyperresponsiveness than females (p < 0.05). Inflammatory indices including bronchoalveolar lavage fluid total cells, neutrophils, and TNF-alpha content were greater in males than in females 6 h following LPS administration (p < 0.05), whereas whole-lung TLR-4 protein levels did not differ among treatment groups, suggesting that differential expression of TLR-4 before or after LPS exposure did not underlie the observed inflammatory outcomes. Gonadectomy decreased airway inflammation in males but did not alter inflammation in females, whereas administration of exogenous testosterone to intact females increased their inflammatory responses to levels observed in intact males. LPS-induced airway hyperresponsiveness was also decreased in castrated males and was increased in females administered exogenous testosterone. Collectively, these data indicate that airway responsiveness in naive mice is influenced by gender, and that male mice have exaggerated airway inflammatory and functional responses to LPS compared with females. These gender differences are mediated, at least in part, by effects of androgens.