Highly Proliferative α-Cell-Related Islet Endocrine Cells in Human Pancreata.

The proliferative response of non-ß islet endocrine cells in response to type 1 diabetes (T1D) remains undefined. We quantified islet endocrine cell proliferation in a large collection of nondiabetic control and T1D human pancreata across a wide range of ages. Surprisingly, islet endocrine cells with abundant proliferation were present in many adolescent and young-adult T1D pancreata. But the proliferative islet endocrine cells were also present in similar abundance within control samples. We queried the proliferating islet cells with antisera against various islet hormones. Although pancreatic polypeptide, somatostatin, and ghrelin cells did not exhibit frequent proliferation, glucagon-expressing α-cells were highly proliferative in many adolescent and young-adult samples. Notably, α-cells only comprised a fraction (∼1/3) of the proliferative islet cells within those samples; most proliferative cells did not express islet hormones. The proliferative hormone-negative cells uniformly contained immunoreactivity for ARX (indicating α-cell fate) and cytoplasmic Sox9 (Sox9Cyt). These hormone-negative cells represented the majority of islet endocrine Ki67+ nuclei and were conserved from infancy through young adulthood. Our studies reveal a novel population of highly proliferative ARX+ Sox9Cyt hormone-negative cells and suggest the possibility of previously unrecognized islet development and/or lineage plasticity within adolescent and adult human pancreata.

ß Cells Persist in T1D Pancreata Without Evidence of Ongoing ß-Cell Turnover or Neogenesis.

Context: The cellular basis of persistent ß-cell function in type 1 diabetes (T1D) remains enigmatic. No extensive quantitative ß-cell studies of T1D pancreata have been performed to test for ongoing ß-cell regeneration or neogenesis. Objective: We sought to determine the mechanism of ß-cell persistence in T1D pancreata. Design: We studied T1D (n = 47) and nondiabetic control (n = 59) pancreata over a wide range of ages from the Juvenile Diabetes Research Foundation Network of Pancreatic Organ Donors with Diabetes via high-throughput microscopy. Intervention and Main Outcome Measures: We quantified ß-cell mass, ß-cell turnover [via Ki-67 and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)], islet ductal association, and insulin/glucagon coexpression in T1D and control pancreata. Results: Residual insulin-producing ß cells were detected in some (but not all) T1D cases of varying disease duration. Several T1D pancreata had substantial numbers of ß cells. Although ß-cell proliferation was prominent early in life, it dramatically declined after infancy in both nondiabetic controls and T1D individuals. However, ß-cell proliferation was equivalent in control and T1D pancreata. ß-cell death (assessed by TUNEL) was extremely rare in control and T1D pancreata. Thus, ß-cell turnover was not increased in T1D. Furthermore, we found no evidence of small islet/ductal neogenesis or α-cell to ß-cell transdifferentiation in T1D pancreata, regardless of disease duration. Conclusion: Longstanding ß-cell function in patients with T1D appears to be largely a result of ß cells that persist, without any evidence of attempted ß-cell regeneration, small islet/ductal neogenesis, or transdifferentiation from other islet endocrine cell types.

Identification of Transthyretin Cardiac Amyloidosis Using Serum Retinol-Binding Protein 4 and a Clinical Prediction Model.

Importance: Transthyretin cardiac amyloidosis (ATTR) is an underrecognized cause of heart failure (HF) in older individuals, owing in part to difficulty in diagnosis. ATTR can result from substitution of valine for isoleucine at codon 122 of the transthyretin (TTR) gene (V122I), present in 3.43% of African American individuals. Objective: To examine whether serum retinol-binding protein 4 (RBP4), an endogenous TTR ligand, could be used as a diagnostic test for ATTR V122I amyloidosis. Design, Setting, and Participants: In this combined prospective and retrospective cohort study performed at a tertiary care referral center, 50 African American patients 60 years or older with nonamyloid HF and cardiac wall thickening prospectively genotyped from September 1, 2014, through December 31, 2015, and a comparator cohort of 25 patients with biopsy-proven ATTR V122I amyloidosis recruited from September 1, 2009, through November 31, 2014, comprised the development cohort. Twenty-seven African American patients and 9 patients with ATTR V122I amyloidosis comprised the validation cohort. Main Outcomes and Measures: Circulating RBP4, TTR, B-type natriuretic peptide (BNP), and troponin I (TnI) concentrations and electrocardiographic, echocardiographic, and clinical characteristics were assessed in all patients. Receiver operating characteristic (ROC) analysis was performed to identify optimal thresholds for ATTR V122I amyloidosis identification. A clinical prediction rule was developed using penalized logistic regression, evaluated using ROC analysis and validated in an independent cohort of cases and controls. Results: Age, sex, and BNP and TnI concentrations were similar between the 25 patients with ATTR V122I amyloidosis (mean [SD] age, 72.2 [7.4] years; 18 male [72%]) and the 50 controls (mean [SD] age, 69.2 [5.7] years; 31 male [62%]). Serum RBP4 concentration was lower in patients with ATTR V122I amyloidosis compared with nonamyloid controls (31.5 vs 49.4 µg/mL, P < .001), and the difference persisted after controlling for potential confounding variables. Left ventricular ejection fraction was lower in patients with ATTR V122I amyloidosis (mean [SD], 40% [14%] vs 57% [14%], P < .001), whereas interventricular septal diameter was higher (mean [SD], 16 [3] vs 14 [2] mm, P < .001). The ROC analysis identified RBP4 as a sensitive identifier of ATTR V122I amyloidosis (area under the curve [AUC] = 0.78; 95% CI, 0.67-0.88). A clinical prediction algorithm composed of RBP4, TTR, left ventricular ejection fraction, interventricular septal diameter, mean limb lead QRS voltage, and grade 3 diastolic dysfunction yielded excellent discriminatory capacity for ATTR V122I amyloidosis (AUC = 0.97; 95% CI, 0.93-1.00), whereas a 4-parameter model, including RBP4 concentration, retained excellent discrimination (AUC = 0.92; 95% CI, 0.86-0.99). The models maintained excellent discrimination in the validation cohort. Conclusions and Relevance: A prediction model using circulating RBP4 concentration and readily available clinical parameters accurately discriminated ATTR V122I amyloidosis from nonamyloid HF in a case-matched cohort. This clinical algorithm may be useful for identification of ATTR V122I amyloidosis in elderly African American patients with HF.

The prevalence and distribution of the amyloidogenic transthyretin (TTR) V122I allele in Africa.

BACKGROUND: Transthyretin (TTR) pV142I (rs76992529-A) is one of the 113 variants in the human TTR gene associated with systemic amyloidosis. It results from a G to A transition at a CG dinucleotide in the codon for amino acid 122 of the mature protein (TTR V122I). The allele frequency is 0.0173 in African Americans. METHODS: PCR-based assays to genotype 2767 DNA samples obtained from participants in genetic studies from various African populations supplemented with sequencing data from 529 samples within the 1000 Genomes Project. RESULTS: The rs76992529-A variant allele was most prevalent (allele frequency 0.0253) in the contiguous West African countries of Sierra Leone, Guinea, Ivory Coast, Burkina Faso, Ghana, and Nigeria. In other African countries, the mean allele frequency was 0.011. CONCLUSIONS: Our data are consistent with a small number of founder carriers of the amyloidogenic TTR V122I (p.Val142Ile) allele in southern West Africa, with no apparent advantage or disadvantage of an allele carrying newborn reaching adulthood. In U.S. African Americans, the allele represents a significant risk for congestive heart failure late in life. If clinical penetrance is similar in African countries with high allele frequencies, then cardiac amyloidosis could also represent a significant cause of heart disease in the elderly in those populations.

Prevalence of the amyloidogenic transthyretin (TTR) V122I allele in 14 333 African-Americans.

BACKGROUND: Transthyretin (TTR) V122I (rs76992529) is one of 111 variants caused by point mutations in the coding sequence of the human TTR gene that are associated with systemic amyloidosis. It results from a G to A transition at a CG dinucleotide in codon 142(122 of the mature protein) of the gene and has been described almost exclusively in people of African descent. Several series have reported allele frequencies from 0.015 to 0.020 in African-Americans. OBJECTIVE: To define more accurately the frequency of the TTR V122I variant allele in the African-American population. METHODS: DNA isolated from blood spots from 1688 New York State African-American newborns was genotyped for the TTR V122I allele. We also compiled new data from the Jackson Heart Study and previously unpublished data from the Dallas Heart Study, plus data from a San Diego "wellness study", providing 15 650 additional allelotypes to those already reported. RESULTS: Among the New York newborns, the TTR V122I allele was present in 65/3376 alleles (allele prevalence 0.0193). The combined available data from all the non-selected African-American cohorts showed the TTR variant allele to be present in 451/26 062 alleles (allele prevalence of 0.0173), slightly but not significantly lower than our previously published estimates. CONCLUSIONS: The allele prevalence for TTR V122I in African-Americans is 0.0173. Of African-Americans under age 65, 3.43% carry at least one copy of the variant amyloidogenic allele.

Epoetin alfa versus darbepoetin alfa in chemotherapy-related anemia.

OBJECTIVE: To review and compare the data concerning the clinical activity of epoetin alfa versus darbepoetin alfa when administered to patients with cancer who are experiencing treatment-related anemia. DATA SOURCES: English-language publications from the MEDLINE database (1990-June 2005), published articles, and meeting abstracts were reviewed. STUDY SELECTION AND DATA EXTRACTION: Relevant data were extracted from published reports and abstracts on studies of humans with cancer who developed treatment-related anemia and were treated with epoetin alfa or darbepoetin alfa. DATA SYNTHESIS: Epoetin alfa and darbepoetin alfa are similar agents with identical indications for treatment of anemia in patients with cancer. Clinical trials have demonstrated that both agents can significantly improve hemoglobin levels, reduce transfusion requirements, and improve quality of life. Epoetin alfa is approved for administration at a dose of 150 units/kg subcutaneously 3 times per week, and darbepoetin alfa is approved for administration at a dose of 2.25 units/kg once a week. Clinical studies have demonstrated that epoetin alfa may be administered at 40,000 units once a week and that darbepoetin alfa may be administered at 200 microg every 2 weeks without loss of efficacy. Cost analysis, based on the average wholesale price of each drug alone administered for 12 weeks at Food and Drug Administration-approved doses, revealed that epoetin alfa is less expensive than darbepoetin alfa. When they are administered in the extended schedules, the cost of darbepoetin alfa is slightly less than that of epoetin alfa. However, the total expense associated with the extended schedule of either agent is further reduced by a reduction in other costs associated with drug administration. CONCLUSIONS: Epoetin alfa and darbepoetin alfa have identical indications for treatment of anemia in patients receiving cancer chemotherapy. Clinical trials have demonstrated similar activities with both agents. Darbepoetin alfa, with a longer half-life, can be administered less frequently, saving costs as well as reducing patient office visits.

Sentinel lymph node mapping and molecular staging in nonsmall cell lung carcinoma.

BACKGROUND: Lymph node (LN) involvement predicts recurrence in patients who have undergone resection of apparently localized nonsmall cell lung carcinoma (NSCLC). Standard detection methods for LN disease have a low sensitivity, and many patients with apparent N0 disease status develop recurrent disease. Molecular techniques can improve the detection of micrometastases, whereas sentinel lymph node (SLN) mapping can indicate which LN may contain micrometastases. These methods, although potentially complementary, have not, to the authors' knowledge, been used together previously. METHODS: The authors used SLN mapping and molecular staging to improve the detection of LN micrometastases in patients with NSCLC. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for cytokeratin-7 (CK7), expressed both in normal lung and in malignant lung, was used to identify tumor-derived material in LN. RESULTS: SLN mapping was performed in 13 patients, with 1-3 SLNs identified in each patient, and sufficient RNA for RT-PCR was obtained in 12 of these 13 patients. Eleven of 12 tumors expressed CK7. Overall, 32 LNs were positive for CK7, including 13 of 21 SLNs. Ten of 11 patients with evaluable SLNs had at least 1 CK7-positive SLN. Routine pathology showed Stage I disease in eight patients, T3N0 disease in one patient, and LN-positive disease in two patients. Of the nine patients with N0 disease according to routine pathology that was evaluable by RT-PCR, eight patients were upstaged by this technique. All patients with positive LN status by routine pathology who were evaluable by RT-PCR analysis had positive RT-PCR results. CONCLUSIONS: LN micrometastases were common in resected NSCLC, including patients with N0 disease according to routine pathology. SLN mapping was useful for identifying disease-containing LNs. This approach may be useful for stratifying histologically N0 patients into higher risk and lower risk groups.

Drop-in, drop-out allele-specific PCR: a highly sensitive, single-tube method.

Allelotyping large numbers of samples by allele-specific polymerase chain reaction (PCR) can be problematic if the DNA samples to be tested are of highly variable concentration. On the one hand, analysis of dilute DNA samples often requires nested PCR to produce a product of sufficient yield to be detectable on ethidium bromide-stained agarose gels. Such two-step assays require additional reagents, are labor-intensive, and have a higher risk of contamination. On the other hand, the specificity of allele-specific PCR assays can be lost at high input DNA concentrations. Large population-based genetic studies using DNA from varied sources would benefit from one-tube assays that could detect mutations in samples over a wide range of concentration. We describe a one-tube nested allele-specific PCR-based assay, in which the input DNA concentration has little effect on the assay's yield or specificity. An assay using this method is highly sensitive and specific, and was used to type several thousand DNA samples, obtained from various sources, for a G to A transition at human transthyretin codon 122. Similar assays could be readily adapted to any high-throughput allelotype assay where input DNA is of highly variable concentration.

Chronic lymphocytic leukemia with prostate infiltration mediated by specific clonal membrane-bound IgM.

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of monoclonal B lymphocytes in the hematopoietic organs. Rarely, CLL cells accumulate in a single atypical site. The mechanism underlying this unusual distribution of CLL cells has not been studied previously. We obtained peripheral blood from five patients having early stage CLL with heavy prostate infiltration. These patients' circulating CLL cells bound strongly in vitro to cultured prostate cell lines PC3, LNCaP, and DU145 and to short-term cultures of fresh prostate cells but not to colon, breast, or bladder cells. CLL cells from patients without prostate infiltration did not bind in vitro to any cell line. Peripheral blood CLL cells from one patient with CLL with heavy prostate infiltration were fused with a mouse-human heteromyeloma line to make hybridomas expressing the same monoclonal IgM as the patient's CLL cells. The hybridoma cells bound specifically to prostate cells. IgM secreted by the hybridoma blocked binding of the patient's CLL cells to prostate cells. Flow cytometry and immunohistochemistry demonstrated that the secreted IgM bound specifically to prostate cells. These results indicate that CLL with atypical prostate infiltration can be mediated by specific surface-bound IgM against an antigen expressed specifically by prostate cells and suggest that a similar mechanism might also apply to cases of CLL with atypical infiltration into other organs.

Mice transgenic for human TTR have the same frequency of renal TTR deposition whether maintained in conventional or specific pathogen free environments.

Mice transgenic for many copies of the human wild type transthyretin gene were bred and maintained in a specific pathogen free environment until twelve months of age. At that time, half the animals were moved to a conventional animal facility. The incidence of both transthyretin and AA amyloid was the same in both groups at two years of age, indicating that in this model, the frequency, extent or nature of TTR-amyloid deposition did not differ significantly between conventional and specific pathogen-free environments.