Engineering CD3/CD137 dual specificity into a DLL3-targeted T-cell engager enhances T-cell infiltration and efficacy against small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICIs) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all SCLC patients are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared to a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

Engineering CD3/CD137 Dual Specificity into a DLL3-Targeted T-Cell Engager Enhances T-Cell Infiltration and Efficacy against Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

Characterizations of a neutralizing antibody broadly reactive to multiple gluten peptide:HLA-DQ2.5 complexes in the context of celiac disease.

In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.

Association of disability level with polypharmacy and potentially inappropriate medication in community dwelling older people.

AIMS: To investigate the influence of disability severity level on polypharmacy and the prescription of potentially inappropriate medication (PIM) using health insurance and long-term care (LTC) insurance claim data. METHODS: Data were obtained from a health-care insurance and long-term care insurance claims database of citizens of Kure city, Japan, in April 2017. Data including age, sex, and drug profile were obtained, and the level of LTC needs was used to measure disability level. Factors associated with polypharmacy (≥5 prescribed drugs) and PIM prescription (≥1 PIM) defined by STOPP-J were analyzed statistically. RESULTS: Among 67,169 people aged ≥65 (mean age 77.2 ± 7.9, male 40.7%), the frequency of polypharmacy increased with age until 85-89 (male 58.3%, female 57.6%) in both genders, and polypharmacy was most prevalent in those at the mildest LTC level (support level: male 68.9%, female 73.7%). PIM prescriptions was also frequent in those with LTC needs. On multiple logistic regression analysis, polypharmacy was significantly associated with older age and LTC needs, and PIM prescription was associated with older age and higher LTC level, suggesting that there is a large difference in prescription according to the person's age and disability level. CONCLUSION: Polypharmacy was prevalent especially in older persons which peaked at the age of 85-89 or at mild disability level, and PIM prescription was prevalent in those with older age and higher care levels. When optimizing polypharmacy or PIM prescription in older patients, healthcare providers should focus on not only age but also disability level.

Melanin-producing medullary thyroid carcinoma with transformation to melanoma: A case report.

Only one case of melanoma arising from melanin-producing medullary thyroid carcinoma (MTC) has been reported previously. In the present study, a second such case was reported and compared with the previous one. The patient was an 86-year-old male who presented with a right anterior neck mass. Ultrasound revealed a nodule measuring 49x48x40 mm in the right lobe of the thyroid. The levels of serum calcitonin (2,298 pg/ml) and carcinoembryonic antigen (CEA; 27.0 ng/ml) were markedly elevated. Aspiration cytology revealed suspected malignant anaplastic thyroid carcinoma and total thyroidectomy without neck nodal dissection was performed. On gross observation, the nodule was well encapsulated, soft, solid and black. Light microscopy indicated that the nodule was composed mainly of large, occasionally huge, pleomorphic cells with a solid or alveolar growth pattern. On immunohistochemistry, these cells were positive for melan-A and S-100 protein, and negative for thyroid transcription factor 1, calcitonin, chromogranin A and CEA. In the subcapsular area, melanin-producing MTC was intimately intermingled with the pleomorphic cells. No primary site of the melanoma was detectable in other organs. At three years after surgery, the patient died due to metastasis of the melanoma to the brain. The previously reported case had no detectable recurrence or distant metastasis up to 11 years after surgery. In comparison with that case, the present case had a similar morphology but the outcome was poorer. Thus, the prognosis of melanoma that transforms from MTC appears to remain uncertain.

Utilization of drugs for the management of cardiovascular diseases at intermediate care facilities for older adults in Japan.

OBJECTIVES: No established approaches exist for the pharmacological management of cardiovascular diseases (CVDs) in residents of long-term care facilities (LTCFs). This study aimed to evaluate the use of drugs for CVD prevention and treatment (CVD-related drugs) in a major type of LTCF in Japan. METHODS: This study included 1318 randomly selected residents at 349 intermediate care facilities for older adults (called Roken). Prescriptions were investigated at admission and two months after admission according to therapeutic categories. Logistic regression was used to identify residents' characteristics that were associated with prescriptions of CVD-related drugs. RESULTS: Prescriptions of all types of drugs and CVD-related drugs decreased in 36 % and 16 % of residents, respectively. Half of the residents received antihypertensives, a quarter received antiplatelets and diuretics, whereas one-tenth received antidiabetics, oral anticoagulants, and lipid-modifying drugs. The prevalence of most of individual drug categories were similar among residents with different physical or cognitive function, except for fewer antihypertensive and lipid-modifying drugs in those with severe cognitive disability. Adjusted analyses for prescriptions at two months after admission revealed that bedridden residents were more likely to be prescribed diuretics but less likely to be prescribed antihypertensives, antiplatelets, or lipid-modifying drugs. Residents with severe cognitive disability were less likely to be prescribed antihypertensives or lipid-modifying drugs. A known history of cardiovascular events was associated with greater use of CVD-related drugs. CONCLUSION: CVD-related drugs were commonly prescribed for Roken residents, including those with low physical and cognitive functions. Deprescribing may contribute to the optimization of pharmacotherapy in LTCF residents.

Prevalence of cytochrome P450-mediated potential drug-drug interactions in residents of intermediate care facilities for older adults in Japan.

AIM: Limited information is available on the prevalence of drug-drug interactions (DDI) in residents of long-term care facilities who often receive multiple drugs. This study aimed to evaluate the prevalence of clinically relevant cytochrome P450-mediated potential DDI in residents of intermediate care facilities for older adults (called Roken) in Japan. METHODS: A nationwide drug utilization study was carried out for Roken residents in 2015 (up to five residents per facility). Potential DDI were identified with an explicit list of drugs that can be involved in clinically relevant cytochrome P450-mediated DDI in Japan. Logistic regression was used to evaluate the association of the number of drugs prescribed with the presence of potential DDI, adjusted for age, sex and long-term care needs level. RESULTS: The study included 1222 residents of 348 Roken who were prescribed two or more active drug substances. The participants who received ≥6 and ≥10 drugs represented 49% and 10% of total participants, respectively. In total, 42 two-drug combinations involving potential DDI were identified in 33 participants (2.7%) - benzodiazepines, proton pump inhibitors, calcium channel blockers and anti-epileptic drugs were frequently involved. The adjusted odds ratios for potential DDI were 2.84 (95% confidence interval 1.15-7.02) or 7.82 (95% confidence interval 2.96-20.70) in residents receiving six to nine drugs or ≥10 drugs, compared with those receiving two to five drugs. CONCLUSIONS: Approximately 3% of Roken residents were at risk for clinically relevant DDI. Reducing the number of drugs prescribed through medication reviews would mitigate the potential risk. Geriatr Gerontol Int 2019; 19: 513-517.

Identifying drug substances of screening tool for older persons' appropriate prescriptions for Japanese.

BACKGROUND: In 2015, the Japan Geriatric Society (JGS) updated "the Guidelines for Medical Treatment and its Safety in the elderly," accompanied with the Screening Tool for Older Persons' Appropriate Prescriptions for Japanese (STOPP-J): "drugs to be prescribed with special caution" and "drugs to consider starting." The JGS proposed the STOPP-J to contribute to improving prescribing quality; however, each decision should be carefully based on medical knowledge. The STOPP-J shows examples of commonly prescribed drug substances, but not all relevant drugs. This research aimed to identify substances using such coding, as a standardized classification system would support medication monitoring and pharmacoepidemiologic research using such health-related information. METHODS: A voluntary team of three physicians and two pharmacists identified possible approved medicines based on the STOPP-J, and matched certain drug substances to the Anatomical Therapeutic Chemical Classification (ATC) and the Japanese price list as of 2017 February. Injectables and externally used drugs were excluded, except for self-injecting insulin, since the STOPP-J guidelines are intended to cover medicines used chronically for more than one month. Some vaccines are not available in the Japanese price list since they not reimbursed through the national health insurance. RESULTS: The ATC 5th level was not available for 39 of the 235 identified substances, resulting in their classification at the ATC 4th level. Furthermore, among 26 combinations, 10 products were matched directly to the ATC 5th level of the exact substances, and others were linked to the ATC representing the combination or divided into multiple substances for classification if the combination was not listed in the ATC. CONCLUSION: This initial work demonstrates the challenge of matching ATC codes and the Japan standard commodity classification codes corresponding to STOPP-J substances. Since coding facilitates database analysis, the proposed drug list could be applied to research using large databases to examine prescribing patterns in patients older than 75 years or who are frail. Since ATC is not available for some substances, Japanese medicines need the process to be registered in the ATC for an effective screening tool to be developed for STOPP-J.

Screening Tool for Older Persons' Appropriate Prescriptions for Japanese: Report of the Japan Geriatrics Society Working Group on "Guidelines for medical treatment and its safety in the elderly".

AIM: In 2005, the Japan Geriatrics Society published a list of potentially inappropriate medication that was an extract from the "Guidelines for medical treatment and its safety in the elderly 2005." The 2005 guidelines are due for a revision, and a new comprehensive list of potentially inappropriate medications is required. METHODS: A total of 15 diseases, conditions and special areas related to their clinical care were selected. We originated clinical questions and keywords for these 15 areas, carried out a systematic review using these search criteria, and formulated guidelines applying the Grading of Recommendations Assessment, Development and Evaluation system advocated by Minds2014. If we did not find good evidence despite the drug being clinically important, we looked for evidence of efficacy and for disease-specific guidelines, and incorporated them into our guidelines. RESULTS: We selected 2098 articles (140 articles per area), and extracted another 186 articles through a manual search. We further added guidelines based on disease entity and made two lists, one of "drugs to be prescribed with special caution" and the other of "drugs to consider starting," primarily considering individuals aged 75 years or older or those who are frail or in need of special care. CONCLUSIONS: New lists of potentially inappropriate medications and potential prescribing omissions called "Screening Tool for Older Person's Appropriate Prescriptions for Japanese" were constructed. We anticipate that future studies will highlight more evidence regarding the safety of high-quality drugs, further improving the provision of appropriate medical care for the elderly. Geriatr Gerontol Int 2016: 16: 983-1001.

Urinary N-telopeptide and Rate of Bone Loss Over the Menopause Transition and Early Postmenopause.

The purpose of this study was to assess the ability of urinary N-telopeptide (U-NTX) to gauge rate of bone loss across and after the menopause transition (MT). U-NTX measurement was measured in early postmenopause in 604 participants from the Study of Women's Health Across the Nation (SWAN). We examined the association between U-NTX and annualized rates of decline in lumbar spine and femoral neck bone mineral density (BMD) across the MT (1 year before the final menstrual period [FMP] to time of U-NTX measurement), after the MT (from time of U-NTX measurement to 2 to 4 years later), and over the combined period (from 1 year before FMP to 2 to 4 years after U-NTX measurement). Adjusted for covariates in multivariable linear regression, every standard deviation (SD) increase in U-NTX was associated with 0.6% and 0.4% per year faster declines in lumbar spine and femoral neck BMD across the MT; and 0.3% (lumbar spine) and 0.2% (femoral neck) per year faster declines over the combined period (across and after the MT) (all p < 0.01). Each SD increase in U-NTX was also associated with 44% and 50% greater risk of fast bone loss in the lumbar spine (defined as BMD decline in the fastest 16% of the distribution) across the MT (p < 0.001, c-statistic = 0.80) and over the combined period (across and after the MT) (p = 0.001, c-statistic = 0.80), respectively. U-NTX measured in early postmenopause is most strongly associated with rates of bone loss across the MT, and may aid early identification of women who have experienced fast bone loss during this critical period. © 2016 American Society for Bone and Mineral Research.

Quantifying the Balance Between Total Bone Formation and Total Bone Resorption: An Index of Net Bone Formation.

CONTEXT: Bone gain vs loss across the skeleton loss depends on the balance between total bone formation and total bone resorption. OBJECTIVE: The objective of the study was to determine whether resorption and formation markers can be combined to gauge net bone formation across the skeleton. DESIGN: The study included a cohort followed up across menopause transition (Study of Women's Health Across the Nation). SETTING AND PARTICIPANTS: Community-dwelling women, 42-52 years old, premenopausal or early perimenopausal at baseline, participated in the study. OUTCOME: The study included the following measures: 1) bone balance index (BBI) created by estimating the relationship between resorption (urinary N-telopeptide) and formation (osteocalcin) markers when the total formation equals the total resorption in 685 women with stable bone mineral density (BMD) (>5 y before the final menstrual period [FMP]) and applying this relationship to measured bone turnover markers in 216 women beginning to lose bone (≤2 y from FMP); and 2) annualized percentage declines over the following 3-4 years in the lumbar spine (LS) and femoral neck (FN) BMD. RESULTS: Adjusted for covariates, the BBI was greater (more favorable) in women with a greater body mass index (P = .03) and lower (less favorable) in women closer to the FMP (P = .007). Each SD decrement in BBI was associated with 0.27%/y faster LS BMD decline (P 0.04) and a 38% higher odds of faster-than-average loss of LS bone mass (P = .008, c-statistic 0.76). BBI was not associated with decline in FN BMD. Urinary N-telopeptide alone was not associated with either LS or FN BMD decline. CONCLUSIONS: An index that quantifies net bone formation vs resorption can be created from bone turnover markers and may help identify individuals at high risk for LS bone loss.

The State of Health in Older Adults in Japan: Trends in Disability, Chronic Medical Conditions and Mortality.

Both life expectancy and healthy life expectancy in Japan have been increasing and are among the highest in the world, but the gap between them has also been widening. To examine the recent trends in old age disability, chronic medical conditions and mortality in Japan, we retrospectively analyzed three nationally representative datasets: Comprehensive Survey of Living Conditions (2001-2013), Patient Survey (1996-2011) and Vital Statistics (1995-2010). We obtained the sex- and age-stratified trends in disability rate, treatment rates of nine selected chronic medical conditions (cerebrovascular diseases, joint disorders, fractures, osteoporosis, ischemic heart disease, diabetes mellitus, hypertension, pneumonia and malignant neoplasms), total mortality rate and mortality rates from specific causes (cerebrovascular diseases, heart diseases, pneumonia and malignant neoplasms) in both sexes in four age strata (65-69, 70-74, 75-79, 80-84 years). Disability rates declined significantly in both sexes. Treatment rates of all selected medical conditions also decreased significantly, except for fractures in women and pneumonia. Both total mortality rate and cause-specific mortality rates decreased in both sexes. We concluded that the recent decline in disability rates, treatment rates of chronic medical conditions and mortality rates points toward overall improvement in health conditions in adults over the age of 65 years in Japan. Nonetheless, considering the increase in the number of older adults, the absolute number of older adults with disability or chronic medical conditions will continue to increase and challenge medical and long-term care systems.

Development of a simple screening test for sarcopenia in older adults.

AIM: To develop a simple screening test to identify older adults at high risk for sarcopenia. METHODS: We studied 1971 functionally independent, community-dwelling adults aged 65 years or older randomly selected from the resident register of Kashiwa city, Chiba, Japan. Data collection was carried out between September and November 2012. Sarcopenia was defined based on low muscle mass measured by bioimpedance analysis and either low muscle strength characterized by handgrip or low physical performance characterized by slow gait speed. RESULTS: The prevalence of sarcopenia was 14.2% in men and 22.1% in women. After the variable selection procedure, the final model to estimate the probability of sarcopenia included three variables: age, grip strength and calf circumference. The area under the receiver operating characteristic curve, a measure of discrimination, of the final model was 0.939 with 95% confidence interval (CI) of 0.918-0.958 for men, and 0.909 with 95% CI of 0.887-0.931 for women. We created a score chart for each sex based on the final model. When the sum of sensitivity and specificity was maximized, sensitivity, specificity, and positive and negative predictive values for sarcopenia were 84.9%, 88.2%, 54.4%, and 97.2% for men, 75.5%, 92.0%, 72.8%, and 93.0% for women, respectively. CONCLUSIONS: The presence of sarcopenia could be detected using three easily obtainable variables with high accuracy. The screening test we developed could help identify functionally independent older adults with sarcopenia who are good candidates for intervention.

C-reactive protein, bone strength, and nine-year fracture risk: data from the Study of Women's Health Across the Nation (SWAN).

Higher levels of C-reactive protein (CRP), an inflammatory marker, are associated with increased fracture risk, although previous studies on CRP and bone mineral density (BMD) have yielded conflicting results. We aimed to test the hypotheses that composite indices of femoral neck strength relative to load, which are inversely associated with fracture risk, would also be inversely associated with CRP, and would explain part of the association between CRP and fracture risk. We analyzed data from a multisite, multiethnic prospective cohort of 1872 community-dwelling women, premenopausal or early perimenopausal at baseline. Femoral neck composite strength indices in three failure modes were calculated using dual-energy X-ray absorptiometry (DXA)-derived femoral neck width (FNW), femoral neck axis length (FNAL), femoral neck BMD and body size at baseline, as BMD*FNW/weight for compression strength, BMD*(FNW)(2) /(FNAL*weight) for bending strength, and BMD*FNW*FNAL/(height*weight) for impact strength. Incident nondigital, noncraniofacial fractures were ascertained annually over a median follow-up of 9 years. In analyses adjusted for age, race/ethnicity, diabetes, menopause transition stage, body mass index, smoking, alcohol use, physical activity, medications, prior fracture, and study site, CRP was associated inversely with each composite strength index (0.035-0.041 SD decrement per doubling of CRP, all p < 0.001), but not associated with femoral neck or lumbar spine BMD. During the follow-up, 194 women (10.4%) had fractures. In Cox proportional hazards analyses, fracture hazard increased linearly with loge (CRP), only for CRP levels ≥ 3 mg/L. Addition of femoral neck or lumbar spine BMD to the model did not attenuate the CRP-fracture association. However, addition of any of the composite strength indices attenuated the CRP-fracture association and made it statistically nonsignificant. We conclude that fracture risk increases with increasing CRP, only above the threshold of 3 mg/L. Unlike BMD, composite strength indices are inversely related to CRP levels, and partially explain the increased fracture risk associated with inflammation.

Diabetes and femoral neck strength: findings from the Hip Strength Across the Menopausal Transition Study.

CONTEXT: Diabetes mellitus is associated with increased hip fracture risk, despite being associated with higher bone mineral density in the femoral neck. OBJECTIVE: The objective of the study was to test the hypothesis that composite indices of femoral neck strength, which integrate dual-energy x-ray absorptiometry derived femoral neck size, femoral neck areal bone mineral density, and body size and are inversely associated with hip fracture risk, would be lower in diabetics than in nondiabetics and be inversely related to insulin resistance, the primary pathology in type 2 diabetes. DESIGN: This was a cross-sectional analysis. SETTING AND PARTICIPANTS: The study consisted of a multisite, multiethnic, community-dwelling sample of 1887 women in pre- or early perimenopause. OUTCOME MEASUREMENTS: Composite indices for femoral neck strength in different failure modes (axial compression, bending, and impact) were measured. RESULTS: Adjusted for age, race/ethnicity, menopausal stage, body mass index, smoking, physical activity, calcium and vitamin D supplementation, and study site, diabetic women had higher femoral neck areal bone mineral density [+0.25 sd, 95% confidence interval (CI) (+0.06, +0.44) sd] but lower composite strength indices [-0.20 sd, 95% CI (-0.38, -0.03) sd for compression, -0.19 sd, 95% CI (-0.38, -0.003) sd for bending, -0.19 sd, 95% CI (-0.37, -0.02) sd for impact] than nondiabetic women. There were graded inverse relationships between homeostasis model-assessed insulin resistance and all three strength indices, adjusted for the same covariates. CONCLUSIONS: Despite having higher bone density, diabetic women have lower indices of femoral neck strength relative to load, consistent with their documented higher fracture risk. Insulin resistance appears to play an important role in bone strength reduction in diabetes.

Comparison of pituitary-adrenal responsiveness between insulin tolerance test and growth hormone-releasing peptide-2 test: a pilot study.

Insulin tolerance test (ITT) is the gold standard for assessing the hypothalamic-pituitary-adrenal (HPA) function. GH-releasing peptide (GHRP)-2, which has a strong GH-stimulating activity, is useful for diagnosing GH deficiency as well as ITT. Additionally, GHRP-2 is also known to activate HPA axis. There have been no comparative studies of pituitary-adrenal responsiveness between GHRP-2 test and ITT in patients with hypothalamic/pituitary disease. To assess whether GHRP-2 test could be an alternative to ITT for diagnosing HPA axis failure, both ITT and GHRP-2 test were performed in 15 patients suspected of hypopituitarism. A 100mug dose of GHRP-2 was administered intravenously and plasma ACTH and serum cortisol concentrations were measured. In ITT, a peak cortisol value over 18mug/dl is considered normal. Nine patients were diagnosed as HPA axis failure by ITT. Their median peak cortisol in GHRP-2 test was 11.4mug/ml. In 6 patients diagnosed as normal HPA axis status by ITT, their median peak cortisol in response to GHRP-2 test was 21.4mug/dl, significantly higher (p=0.0032) than seen in patients diagnosed as HPA axis failure. There was a strong correlation between the peak cortisol in GHRP-2 test and ITT (r=0.817; p<0.0001). When the cut-off value for the peak cortisol in GHRP-2 test was set to 13-14mug/dl for diagnosing HPA axis failure, the specificity and sensitivity were 100% and 88.9%, respectively. Although further studies that include normal subjects are needed, these preliminary results suggest the possibility that GHRP-2 test may be an alternative to ITT for assessing HPA axis function.

Effective screening method of agonistic diabodies based on autocrine growth.

Agonistic diabodies that mimic the function of natural ligands are expected to increase the value of therapeutic antibodies. We have developed a method that detects agonistic diabodies based on their ability to transduce growth signals through receptors, thereby permitting cytokine-independent growth of BaF/3-derived cytokine-dependent cells. Retrovirus-mediated expression of the diabody in cytokine-dependent cells was followed by selection of clones for growth in the absence of cytokine. A diabody library derived from splenocytes of human Mpl immunized mice was constructed. Infection of cells with viral particles led to the isolation of over 500 autonomously growing clones whose cultured supernatants showed agonistic activities against Mpl. Genome-integrated diabodies were cloned; representative clone AB317 showed agonistic activities as potent as a natural ligand and cross-reactive against mouse Mpl.

Reporter expression, induced by a growth hormone promoter-driven Cre recombinase (rGHp-Cre) transgene, questions the developmental relationship between somatotropes and lactotropes in the adult mouse pituitary gland.

This report describes the development and validation of the rGHp-Cre transgenic mouse that allows for selective Cre-mediated recombination of loxP-modified alleles in the GH-producing cells of the anterior pituitary. Initial screening of the rGHp-Cre parental line showed Cre mRNA was specifically expressed in the anterior pituitary gland of adult Cre+/- mice and cephalic extracts of e17 Cre+/- fetuses. Heterozygote rGHp-Cre transgenic mice were crossbred with Z/AP reporter mice to generate Cre+/-,Z/AP+/- offspring. In this model system, the GH promoter-driven, Cre-mediated recombination of the Z/AP reporter leads to human placental alkaline phosphatase (hPLAP) expression that serves to mark cells that currently produce GH, in addition to cells that would have differentiated from GH cells but currently do not express the GH gene. Double immunocytochemistry of adult male and female Cre+/-,Z/AP+/- pituitary cells revealed the majority (approximately 99%) of GH-producing cells of the anterior pituitary also expressed hPLAP, whereas ACTH-, TSH-, and LH-producing cells were negative for hPLAP, confirming previous reports that corticotropes, thyrotropes, and gonadotropes develop independently of the somatotrope lineage. A small subset (approximately 10%) of the prolactin-producing cells was positive for hPLAP, consistent with previous reports showing lactotropes can arise from somatotropes during pituitary development. However, the fact that 90% of prolactin-producing cells were negative for hPLAP suggests that the majority of lactotropes in the adult mouse pituitary gland develop independently of the somatotrope lineage. In addition to developmental studies, the rGHp-Cre transgenic mouse will provide a versatile tool to study the role of a variety of genes in somatotrope function and neoplastic transformation.