Cytological factors that support nonparallel secretion of luteinizing hormone and follicle-stimulating hormone during the estrous cycle

Gonadotropes from cycling female rats were studied to investigate possible mechanisms for the nonparallel release of LH and FSH. The percentages of total gonadotropes increased from 14% during estrus (E) to 18% by diestrous day 2. More of these cells became multihormonal on the morning of proestrus (P; from 46% during diestrus to 69%). Since LH-containing cells increased from 7% at E to 13.3% during early proestrus, this suggests that monohormonal FSH cells may have contributed by synthesizing LH. Gonadotrope cell areas were greatest just before the LH surge (P 1600 h). Microdensitometric measurements demonstrated that the amount and density of immunoperoxidase stain for either gonadotropin subunit were highest during the midafternoon of P. Interestingly, the amount of stain for LH continued to increase during the LH surge, suggesting that the stain had detected newly synthesized LH beta. At the same time, the average density of the LH beta stain decreased. In contrast, the amount, concentration, and density of stain for FSH beta increased during the afternoon of P and decreased during late P and early E. The percentages of granules that contained immunogold stains for only LH or FSH (monohormonal granules) at P 1600-P 1700 h were 3-4 times higher than those in diestrous rats. The percentages of monohormonal LH granules declined during the proestrous surge, whereas percentages of monohormonal FSH granules declined during the first rise (P 1900 h) and after the second rise in serum FSH (E 0800 h). Finally, the average number of gold particles per micron 2 granule area rose over the value in diestrous rats during P 1600-P 1700 h. These studies suggest that multihormonal gonadotropes support nonparallel gonadotropin release by changing the rate of subunit packaging and transit in the Golgi complex.     

Phenotypic and molecular heterogeneity in Philadelphia chromosome-positive acute leukemia

Philadelphia chromosome-positive (Ph1) acute leukemia is a heterogeneous subset of acute leukemia with a poor prognosis. We studied five patients to determine the potential for phenotypic and molecular heterogeneity. Cellular characterization studies included light myeloperoxidase (L-MPO), terminal deoxynucleotidyl transferase (TdT), ultrastructural MPO (U-MPO), and immunophenotyping by flow cytometry using T11, T3, T4, T8, Leu 1, B1, Leu 12, HLA-DR (la), CALLA (J5), OKM1, My4, My7, My8, My9, and My10. DNA was analyzed for rearrangements of the breakpoint cluster region (bcr), immunoglobulin heavy chain, joining region (JH), immunoglobulin kappa light chain constant region (C kappa), and T cell receptor (TcR beta). RNA dot blots were hybridized by using molecular probes for MPO and TdT. We found that four of five cases were acute mixed-lineage leukemia (AMLL). One patient had acute unclassifiable leukemia. Of the four patients classified as having AMLL, three showed myeloid and lymphoid features, with one patient showing myeloid, T cell, and B cell features. The last case showed T cell and B cell features only. In one patient MPO/RNA was positive in spite of insufficient L-MPO or U-MPO to diagnose acute myelogenous leukemia (AML), thereby suggesting significant MPO gene expression before the production of sufficient MPO protein to meet the French-American-British criteria for AML. Three of the five patients showed rearrangement of bcr (cases 1, 2, and 5). Studies of these five patients support the concepts of molecular and phenotypic heterogeneity in Ph1 acute leukemia, demonstrate a high incidence of AMLL in this subset of acute leukemia, and support the use of lineage-associated molecular probes to define lineage at an earlier stage than previously possible.     

Castration induces time-dependent changes in the follicle-stimulating hormone beta-subunit messenger ribonucleic acid-containing gonadotrope cell population

We have previously observed 3- to 10-fold increases in pituitary LH beta-subunit mRNA levels in the rat 28 days after castration. These changes correlate with increases in percentages and areas of cells that bear the LH beta mRNA and with the amount of label for mRNA per cell. In contrast, FSH beta mRNA levels increase 2.5- to 4-fold 7-14 days after castration, decline to near-intact levels 28 days postcastration, and rise 4.5-fold by 96 days postcastration. The purpose of this study was to determine morphological correlates of these changes in FSH beta mRNA levels. Dispersed pituitary cells from intact and castrated rats were analyzed for FSH beta and LH beta mRNAs and protein by in situ hybridization techniques and immunocytochemistry, respectively. In intact animals over 79% of pituitary cells labeled for FSH beta mRNA were small (area less than 150 microns 2). However, 7 days after castration, the average area of labeled cells increased 4-fold (80% were over 200 microns 2 in area), without a significant change in percentages of FSH beta mRNA-containing cells. The amount of mRNA per cell (as measured by area of label per cell) increased 6-fold. Fourteen days after castration, the average area of cells containing FSH beta mRNA decreased to 2 times that in intact rats (48% were greater than 200 microns 2). The percentage of labeled cells increased from 11% (intact) to 20%. Furthermore, the dual labeling studies showed that 37% of these FSH cells were monohormonal (detected by FSH beta mRNA, but not LH beta antigen) compared with 23% in intact rats. At this same time, the FSH cells exhibited a decrease in the amount of mRNA per cell. In 21- to 84-day castrates, average areas of FSH beta mRNA remained at 2-2.5 times the areas of cells from intact rats. In addition, 21 days after surgery the percentages of labeled cells and amount of FSH beta mRNA per cell declined to those in intact rats. A greater proportion was multihormonal (only 15% expressed FSH beta mRNA but not LH beta antigens). At 84 days there were 2-fold increases in the percentages of labeled cells and the density of label, which correlate with the recovery in mRNA levels assayed at 96 days. Thus, factors that contribute to the early rise in FSH beta mRNA include increases in the amount of mRNA per cell, which coincides with increased cell area.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantification of morphological changes in pituitary corticotropes produced by in vivo corticotropin-releasing factor stimulation and adrenalectomy

Physiological and morphological changes produced by corticotropin-releasing factor (CRF) stimulation in vivo were studied in pituitaries immunocytochemically stained for ACTH. After 48-h ip minipump infusions of 10 or 50 ng/min CRF, serum ACTH levels were increased significantly over values in control groups that included both intact rats and rats exposed to sham abdominal surgery. Correlative morphological changes included a striking increase in corticotrope cell area. This was coupled with an apparent decrease in the percentage of stained cells, probably due to degranulation. The cellular responses were similar to those after adrenalectomy described previously by us and others. Therefore, in a parallel study, additional groups of rats were adrenalectomized and studied 24 and 48 h after the surgery. Even greater changes in serum ACTH, corticotrope cell area, and percentages were observed in the adrenalectomized rats. The difference between the CRF-infused and adrenalectomized groups was probably due to the lack of corticosterone feedback in the latter group. Among the control groups, there were no differences between intact rats and rats exposed to sham abdominal surgery. Rats subjected to sham adrenalectomy, however, showed corticotrope responses similar to those of CRF-infused rats, except that the cells were more densely stained. The present studies thus show dramatic changes in ACTH cell area, extent of staining, and percentages after in vivo CRF stimulation. In all of the experimental groups, an excellent correlation existed between serum ACTH levels and the degree of the morphological changes in the corticotropes.     

Purpura fulminans and anticardiolipin antibodies in a patient with Grave's disease

We describe a patient with Grave's discase who developed purpura fulminans and who was found to have anticardiolipin antibodies after being started on propylthiouracil (PTU). We discuss the potential role of the antiphospholipid antibody in this woman's presentation, and its association to both PTU and autoimmune thyroid disease.     

Molecular heterogeneity in acute leukemia lineage switch

Six cases of acute leukemia that underwent lineage switch from acute lymphocytic leukemia to acute myelogenous leukemia are reported. The mean age of the patients was 24 years, time to conversion was 36 months, and survival after conversion was only 3 months. Of the three cases which showed abnormal metaphases at both diagnosis and conversion, two (cases 2, 5) showed related cytogenetic abnormalities, and the third showed (case 3) independent chromosomal changes. Molecular analysis for immunoglobulin heavy chain and T-cell receptor beta chain genes showed that five of the six cases had rearrangement of at least one of these lymphoid associated genes at conversion to acute myelogenous leukemia. The single case (case 3) in which there were no lymphoid gene rearrangements at conversion was also the only case in which independent karyotypic abnormalities at diagnosis and conversion were demonstrated. Our findings suggest that lineage switch can represent either relapse of the original clone with heterogeneity at the molecular level or the emergence of a second new leukemic clone without molecular heterogeneity.     

Efficacy of sulfadoxine/pyrimethamine in the treatment of uncomplicated Plasmodium falciparum malaria in Republic of Congo

Congo is facing frequent failures of treatment of Plasmodium falciparum malaria with chloroquine (CQ), which is still recommended and used as a first-line drug. In Pointe-Noire and Brazzaville, the two largest cities that contain approximately 60% of the population of Congo, we compared the efficacy of CQ versus sulfadoxine/pyrimethamine (SP) for treatment of uncomplicated malaria in children 6-59 months old (mean = 33 months) using the standard World Health Organization (WHO) 14-day in vivo test in two phases between 1999 and 2002. Patients enrolled were randomly assigned to receive SP (25 mg/kg of sulfadoxine and 1.25 mg/kg of pyrimethamine) or CQ (25 mg/kg). In the first phase of the study, 46 patients were assigned to the CQ (n = 23) or SP (n = 23) groups in Pointe-Noire and 52 children were assigned to the CQ (n = 26) or to SP (n = 26) groups in Brazzaville. Results were interpreted according to the WHO lot quality assurance sampling method, and treatment failure rates for SP versus CQ were < 25% versus > 25% in both cities. In the second phase of the study, we accurately determined the actual proportion of treatment failures for SP in Brazzaville. Thus, in 75 of the 80 children enrolled and followed-up until day 14, no clinical or parasitologic failure was recorded and no serious adverse reaction was observed. Since the CQ treatment failure rate exceeds the unacceptable upper limit, SP seems well to be an appropriate alternative for the first-line treatment of uncomplicated P. falciparum malaria, at least in the settings of the present study.     

Prevalence of Hypertension in Patients with Type II Diabetes in Referral versus Primary Care Clinics

We compared the prevalence of hypertension in patients with non–insulin-dependent diabetes mellitus (NIDDM) in referral and primary care practices using definitions of The Fifth Report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure (JNC-V), while controlling for other risk factors such as hypertension, obesity, smoking, and age. Patients (n = 1443) were enrolled consecutively from a large referral practice at the Jackson Diabetes Center and four primary care clinics in the vicinity. Blood pressures were measured at three clinic visits after a 5-min rest in a sitting position using a standard clinical sphygmomanometer. Charts were reviewed to determine diabetes duration, insulin usage, height, weight, smoking history, use of antihypertensive and oral hypoglycemic medications, socioeconomic status, and race. Patients were classified as hypertensive based on JNC-V definitions or if they were on antihypertensive medication. Hypertension was termed uncontrolled if blood pressure was JNC-V Stage 2 or higher while on antihypertensive medication.

Seventy-eight percent of referral clinic and 55% of primary care clinic patients had either JNC-V State 1 or higher hypertension or were on antihypertensive medication. Actual blood pressures indicated that more patients had JNC-V Stage 1 (mild) or higher hypertension in referral compared to primary care clinics (62% versus 48% p = 0.01) but fewer had JNC-V Stage 2 or higher (moderate-severe) hypertension (12% versus 19% p = 0.002). Patients seen in the referral clinic were significantly more likely to have greater age, greater duration of diabetes, higher insulin dosage, longer smoking history, antihypertensive medication, and live outside the metropolitan area. By logistic regression, the odds of hypertension were significantly increased with age (OR 1.51/decade), BMI greater than 27 (OR 2.17), diabetes duration (OR 1.04/year), and insulin dosage (OR 1.74/U/kg). Current smoking and attending a referral clinic were not significantly related. The odds of moderate-severe hypertension were significantly increased with age (OR 1.23/decade), decreased by attending a referral clinic (OR 0.45), and not significantly related to other confounders in the model.

The prevalence of hypertension among patients with NIDDM was higher in referral than primary care clinics. The higher prevalence in the referral practice can be accounted for by the greater severity of associated risk factors in the referral practice patients; however, most patients will be diagnosed and treated for hypertension prior to referral. More patients in the referral practice were on hypertensive medication, which lowered the stage or severity of hypertension but still not to the normal range. The results suggest that the primary detection of hypertension in patients with type II diabetes resides with the primary care physician. Management of hypertension will require both a delineation and acceptance of responsibilities between the primary care physician and diabetes specialists.