The varying effects of a range of preservatives on Myoviridae and Siphoviridae bacteriophages formulated in a semi-solid cream preparation.

Bacteriophages may be formulated into semi-solid bases for therapeutic delivery. This work investigated the effects of a range of preservatives on the viability of Myoviridae and Siphoviridae bacteriophages when these were formulated into a standard semi-solid cream base. The six preservatives tested included: benzoic acid (0·1%), chlorocresol (0·1%), combination hydroxybenzoates (propyl 4-hydroxybenzoates with methyl 4-hydroxybenzoates) (0·1%), methyl 4-hydroxybenzoate (0·08%), 2-phenoxyethanol (1%) and propyl 4-hydroxybenzoate (0·02%). These were each formulated into cetomacrogol cream aqueous to generate six individual semi-solid bases into which Myoviridae and Siphoviridae bacteriophages were added and tested for stability. Optimal bacteriophage stability was seen when the preservative chlorocresol was used. Bacteriophage in the acidic benzoic acid were the least stable, resulting in complete loss of viability after 4-5 weeks. Of the bacteriophages tested, the Myoviridae KOX1 was significantly more stable than the Siphoviridae PAC1 after 91 days in formulations with each of the preservatives. Our results suggest the need for individual testing of specific bacteriophages in pharmaceutical formulations, as their efficacy when exposed to preservatives and excipients in these delivery forms may vary. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages are being increasingly investigated as alternatives to antibiotics. While bacteriophages can be formulated in diverse ways for therapeutic delivery, there has been scant work on how excipients and preservatives in these formulations affect stability of different bacteriophages. We demonstrate that the nature of preservatives in formulations will affect bacteriophage stability, and that in these formulations, viability of bacteriophage differs according to their morphology. Our work highlights the need for individual testing of specific bacteriophages in pharmaceutical formulations, as efficacy when exposed to preservatives and excipients in these delivery forms may vary.

Isolation and characterization of bacteriophage SPI1, which infects the activated-sludge-foaming bacterium Skermania piniformis.

Foaming in activated sludge plants is a worldwide problem commonly caused by proliferation of bacteria of the order Corynebacteriales. These include Skermania piniformis, a filamentous bacterium that has been documented to be a major cause of foaming globally, and particularly in Australian treatment plants. Phage SPI1 is the first phage that was isolated and shown to infect this organism. It targets seven of the nine strains of S. piniformis held in our culture collection, but none of the other 73 mycolata strains of different genera, mostly isolated from wastewater, against which it was tested. Phage SPI1 is a member of the family Siphoviridae and has a circularly permuted dsDNA genome of 55,748 bp with a G+C content of 67.8 mol %. It appears to be obligatorily lytic, with no evidence of genes related to a lysogenic mode of existence.

A novel biopsy method to increase yield of subcutaneous abdominal adipose tissue.

Collection of abdominal subcutaneous adipose tissue (SAT) for research testing is traditionally performed using punch biopsy or needle aspiration techniques, yielding small amounts of very superficial SAT (100-500 mg). Although liposuction techniques can be used to obtain large amounts of SAT, these approaches can compromise the integrity of the adipose tissue. Therefore, we investigated a novel method using a 6-mm Bergström side-cutting biopsy needle to acquire suitable amounts of intact abdominal SAT for multiple complex studies such as flow cytometry, RNA extraction, ex vivo expression of molecular and post-translational protein mediators, and histology. Fifty biopsies were obtained from 29 participants using a Bergström biopsy needle, applying transient manual suction and shearing large pieces of fat within the inner-cutting trochar. Eighteen of the biopsies were performed under ultrasound guidance, whereby we successfully sampled deep SAT (dSAT) from below Scarpa's fascia. The average weight of SAT sampled was 1.5 ± 0.4 g. There was no clinically important bleeding or ecchymosis on the abdominal wall and no infection occurred with this procedure. The 6-mm Bergström biopsy needle yielded substantially more SAT than what has been obtained from superficial procedures and, for the first time, allowed sampling of dSAT by a percutaneous approach.

Adipocyte differentiation is affected by media height above the cell layer.

BACKGROUND: 3T3-L1 cells have been widely used as a model for adipogenesis. However, despite its popularity, differentiation of this cell line has been reported to be inconsistent with low efficiency. OBJECTIVE: To investigate the effect of media height during adipocyte differentiation on lipid accumulation and adipokine secretion in mature adipocytes. METHODS: Three cell lines (3T3-L1, OP9 and ChubS7) were used to test the influence of media volume on adipogenesis. Total lipid content and lipid droplet size and number were quantified. Adipocyte related gene expressions were quantified during the course of differentiation. Secretion of leptin and adiponectin from mature adipocytes were measured using enzyme-linked immunosorbent assays. The influence of oxygen partial pressure on adipogenesis was investigated using three oxygen percentages: 5, 21 and 30%. Insulin sensitivity was measured by insulin inhibition of isoproterenol-induced lipolysis and phosphorylation of insulin receptor substrate-1. RESULTS: A lower media height during adipogenesis increased total lipid accumulation, NEFA release and leptin and adiponectin secretion in mature adipocytes. Insulin sensitivity was not affected by media height during differentiation. CONCLUSION: Media height during adipogenesis was inversely correlated with lipid content in mature adipocytes. To achieve a high lipid content and greater adipokine secretion, it is best to use a low media volume during differentiation.

Vitamin D therapy in patients with primary hyperparathyroidism and hypovitaminosis D.

OBJECTIVE: To determine whether vitamin D repletion of patients with primary hyperparathyroidism (PHPT) and vitamin D deficiency or insufficiency (hypovitaminosis D) has deleterious clinical and/or biochemical effects. DESIGN: Prospective audit of the effect of vitamin D repletion on biochemical data in 56 patients with PHPT. Patients were treated with 50,000 units of vitamin D2 weekly for 8 weeks with biochemical measurements at 5 and 10 weeks, and subsequently after 12 weeks on 800 units of vitamin D3 daily, and in those with hypovitaminosis D after 12 weeks of up to 100 000 units of vitamin D(2) monthly. METHODS: Serum calcium, albumin, phosphorus, 25-OHD, intact parathyroid hormone (PTH) and urine calcium/creatinine (Ca/Cr) ratios were measured before and during vitamin D therapy. RESULTS: Patients treated with 50,000 units of vitamin D2 weekly for 8 weeks resulted in a significant increase in serum 25-OHD levels from 36.4 to 89.4 nmol/l at 5 weeks (P<0.0001) and 88.6 nmol/l at 10 weeks (P<0.0001). There were no significant changes in serum calcium. At 10 weeks, there was a non-significant decrease in serum PTH and in urine Ca/Cr ratios. None of the patients developed any calcium-related adverse events. Subsequently, patients with subnormal 25-OHD levels on 800 units of vitamin D daily were treated for the next 12 weeks with up to 100,000 units of vitamin D2 monthly with normalization of serum 25-OHD in all but 4 patients. CONCLUSION: These data fail to demonstrate any adverse effects of vitamin D repletion in PHPT.

Effect of inappropriate and continuous therapy with alendronate for ten years on skeletal integrity - observations in two elderly patients.

Alendronate is a potent aminobisphosphonate that has been used worldwide to decrease fracture risk in millions of post-menopausal women with and without osteoporosis, men with low bone mass, and in those with glucocorticoid- induced osteoporosis. A recent report of 9 patients with spontaneous atypical non-vertebral fractures during treatment with alendronate for up to 8 yr raised questions suggesting the possibility of severe suppression of bone turnover and resultant susceptibility to fracture. Our recent observations in 2 elderly women with inactive monostotic Paget's disease of bone who had been treated elsewhere continuously for this disease with alendronate for 10 yr at doses overall of 2 and 4 times the osteoporotic dose provided an opportunity to engage in the ongoing controversy over long-term safety of bisphosphonate therapy. Despite such therapy, skeletal integrity was maintained with normal bone densities and the absence of skeletal fractures. These observations do not support the suggestion of deleterious effects with longterm alendronate therapy.

Intravenously administered pamidronate in the treatment of Paget's disease of bone.

OBJECTIVE: To evaluate the effect of intravenously administered pamidronate in patients with Paget's disease of bone. METHODS: We conducted a prospective nonrandomized study and reviewed the related literature. Eighty patients (52 women and 28 men, ranging in age from 53 to 93 years; mean age, 76) were treated with pamidronate intravenously in a total dose of 180 mg during a 6- or 3-week period. All patients had bone scintigraphy and x-ray findings characteristic of Paget's disease of bone. Serum alkaline phosphatase levels were increased in all patients, and findings on fractionation were consistent with a bone origin. Indications for therapy included extensive disease, pagetic deformities, neurologic complications, pagetic pain, and critical areas of involvement. Blood chemistry profile and complete blood cell counts were determined at baseline and after the last dose of pamidronate. Serum calcium levels were determined after the first and second infusions of pamidronate. Patients underwent clinical and biochemical follow-up assessments, with serum alkaline phosphatase measurements, at 2- to 4-month intervals. Many patients who did not have a remission (defined as normalization of serum alkaline phosphatase level) after the first cycle of therapy or who had a relapse with serum alkaline phosphatase levels increased above the normal range after remission were treated with one or more cycles of intravenously administered pamidronate. RESULTS: The mean serum alkaline phosphatase level was 1,051 U/L before therapy and 386 U/L after treatment, a decrease of 63% (P<0.0001). In 50 patients, the serum alkaline phosphatase level declined to normal. Such normalization was noted in 43 of 50 patients (86%) whose baseline alkaline phosphatase was less than 3 times the upper limit of normal (ULN), in 5 of 13 patients (38%) whose baseline alkaline phosphatase was 3 to 6 times the ULN, and in only 2 of 17 patients (12%) whose baseline alkaline phosphatase exceeded 6 times the ULN. Of 30 patients who did not have a remission, 22 received a second course of therapy, 2 of whom had normalization of the serum alkaline phosphatase level. Pamidronate was well tolerated; only nine patients reported a "flu-like" syndrome after the first infusion. Serum calcium levels decreased in many patients (to as low as 8 mg/dL), but no patients were symptomatic. CONCLUSION: With intravenous administration of pamidronate in a dose of 180 mg in patients with Paget's disease of bone, remissions are likely in those whose baseline serum alkaline phosphatase level is <3 times the ULN and unlikely when baseline alkaline phosphatase levels are higher-especially more than 6 times the ULN.

Excretion of sweat and urine pyridinoline crosslinks in healthy controls and subjects with established metabolic bone disease.

Convenient techniques for measuring rates of bone turnover have been developed in recent years with the advent of biochemical markers of bone metabolism. One recent of these techniques is a collection method and quantitative enzyme immunoassay for free pyridinoline crosslinks in human sweat. The concentrations of pyridinoline crosslinks in 5-day sweat collections and first morning void and 24-hour urine collections from healthy subjects and subjects with established metabolic bone disorders were determined. T-scores were higher in the sweat system than in the urine system by up to 10-fold in postmenopausal subjects, women with hyperparathyroidism, and subjects with postmenopausal osteoporosis. For subjects with postmenopausal osteoporosis, receiver-operating characteristic curve analysis yielded areas under the curve of 0.699, 0.629, and 0.520 for sweat pyridinoline, first morning void urine pyridinoline, and 24 hour urine pyridinoline respectively. The areas under the curve of the sweat and first morning void urine measurements were significantly greater (p<0.05) than the 24-hour pyridinoline measurements. Healthy postmenopausal subjects and subjects with postmenopausal osteoporosis were monitored before and during estrogen replacement therapy or alendronate therapy. Sweat pyridinoline values declined by 49.0 +/- 12.4% and 19.4 +/- 19.9% for estrogen and alendronate subjects respectively. We conclude that this non-invasive technique is a sensitive and specific measure of bone resorption and is appropriate as an adjunct to techniques such as bone density and may also be useful in monitoring of response to anti-resorptive therapies.

The effects of event rate and signal probability on children's vigilance.

Using the study of vigilance in adults as their model, the researchers examined task parameters and their interactions in a study of sustained attention in children from a non-clinical population. Two levels of event rate (low and high) were combined with two levels of signal probability (low and high) in 14-minute vigilance tasks in which children viewed small and large squares presented successively on a computer screen. Seven- and eight-year-old children were instructed to press a button whenever a small square appeared. Signal detection analyses were employed, as well as the traditional measures identifying hits, false alarms, and reaction time. The results support the traditional findings in adult tasks: participants performed most accurately and quickly in the high event rate and high probability condition; low probability elicited a more conservative decision-making criterion, a standard characterized by less willingness to risk false alarms; such conservatism increased over the periods of watch; and the vigilance decrement emerged over time. However, the finding that the high event rate condition improved perceptual sensitivity reversed the event rate effect consistently reported in the adult literature. The above findings are discussed in light of both research and clinical implications.

Chronic hypocalcaemia due to selective skeletal resistance to parathyroid hormone.

A 71-year-old man was referred for evaluation of asymptomatic hypocalcaemia dating back at least 20 years. There were no somatic abnormalities and Chvostek and Trousseau signs were negative. Serum total calcium varied from 1.88 to 2.03 mmol/l, albumin 37-44 g/l, phosphate 0.54-1.12 mmol/l and ionized calcium 1-1.13 mmol/l. Serum intact PTH levels were 69 and 55 ng/l (10-65), 25-OHD was 40 nmol/l (2.25-107.5) and 1,25-(OH)2D was 54.6 nmol/l (39-156). Serum and urine magnesium and creatinine clearance were normal. Twenty-four-hour urine calcium was 2.15 mmol and calcium/creatinine ratio 0.07. TM phosphate (maximal rate of tubular reabsorption of phosphate in mmol/l glomerular filtrate (GF)) was 0.84 mmol/l GF (0.80-1.34). Bone formation and resorption markers were normal. Bone mineral densities measured by dual-energy X-ray absorptiometry (DEXA) were within normal limits at the hip, forearm and lumbar spine. Infusion of 200 units of synthetic 1-34 PTH was associated with a rise in urinary cyclic AMP from 43 mmol/l GF to 344 mmol/l GF and TM phosphate fell from 0.93 to 0.76 mmol/l GF; 1-34 PTH infusions of 300 units twice daily for 5 days were associated with an increase in serum 1,25-(OH)2D from 80.6 to 114.4 pmol/l but no increase in serum calcium. This is a most unusual case of chronic hypocalcaemia similar to that reported by Frame et al. resulting from isolated skeletal resistance to PTH that is not related to renal insufficiency, osteomalacia or a magnesium-deficient state. These two cases appear to represent a new variant of pseudohypoparathyroidism ?type III.

Nelson's syndrome due to an intracavernous corticotropin-secreting adenoma.

OBJECTIVE: To report the first case of Nelson's syndrome due to an ectopic intracranial corticotropin-secreting tumor arising entirely within the cavernous sinus. METHODS: We present a case report of Nelson's syndrome with clinical, laboratory, and radiologic features throughout a 25-year period. RESULTS: A 54-year-old woman had been treated for Cushing's disease with bilateral adrenalectomy in 1971. Subsequently, Nelson's syndrome developed, and she had severe generalized hyperpigmentation and substantially increased plasma corticotropin levels. In 1976, she underwent a transsphenoidal hypophysectomy. Postoperatively, despite the development of panhypopituitarism and diabetes insipidus, she remained hyperpigmented and had persistently increased plasma corticotropin levels. Throughout the years, efforts to identify the site of the corticotropin-secreting tumor were unsuccessful until 1988, when magnetic resonance imaging revealed a mass in the right cavernous sinus; subsequently, petrosal sinus cannulation corroborated the intracavernous source of excess corticotropin. Cobalt-60 gamma knife radiotherapy in 1992 was followed by a clinical and hormonal response 4 1/2 years later. CONCLUSION: This report describes only the second reported case of an intracranial corticotropin-secreting tumor arising entirely within the cavernous sinus and the first such case associated with Nelson's syndrome. Although rare, the possibility of an ectopic intracranial or extracranial pituitary adenoma should be considered in patients with pituitary hypersecretion without clear-cut intrasellar abnormalities or those with no response to surgical resection of the pituitary gland.

Neutralization of tumor necrosis factor activity shortly after the onset of dendritic cell hematopoiesis reveals a novel mechanism for the selective expansion of the CD14-dependent dendritic cell pathway.

The CD14-dependent and -independent dendritic cell (DC) pathways are instituted simultaneously when CD34(+) progenitor cells are treated with granulocyte-macrophage colony-stimulating factor (GM-CSF)/tumor necrosis factor (TNF) +/- stem cell factor (SCF) (GTS). If TNF activity is neutralized within 48 hours of cytokine exposure, DC development is halted and myelogranulocytic hematopoiesis takes place. In this study, we show that disruption of TNF activity at a later time point produced a distinct alteration within the DC system. Instead of downregulating DC development, treatment of GTS cultures with antibodies to TNF (anti-TNF) on day 3 provoked the selective expansion of the CD14-dependent (monocyte) DC pathway from progenitor cell populations lacking CD14 and CD1a. After an initial decrease in proliferation, anti-TNF produced a rebound in cell growth that yielded intermediate myeloid progenitors exhibiting CD14-dependent DC differentiation potential and CD14(+)CD1a+ DC precursors. Cultures enriched in CD14-dependent DCs were more potent stimulators of a mixed leukocyte reaction, compared with control GTS cultures containing both types of DCs. The intermediate progenitors expanded in the presence of anti-TNF were CD115(+)CD33(+)DR+, long-lived, and displayed clonogenic potential in methylcellulose. When exposed to the appropriate cytokine combinations, these cells yielded granulocytes, monocytes, and CD14-dependent DCs. Antigen-presenting function was acquired only when DC maturation was induced from these myelodendritic progenitors with GM-CSF + interleukin-4 or GTS. These studies show a novel mechanism by which TNF regulates the DC system, as well as providing a strategy for the amplification of the CD14-dependent DC pathway from immature progenitors. Although TNF is required to ensure the institution of DC hematopoiesis from CD34(+) progenitor cells, its activity on a later progenitor appears to limit the development of CD14-dependent DCs.

Osteoporosis update.

Osteoporosis is a potentially devastating disease affecting millions of Americans with more than 1.5 million fractures annually at a cost that is projected to be more than 60 billion dollars by the year 2020. Heightened patient awareness and education regarding risk factors, especially of behavioral and nutritional factors, should help in prevention particularly when directed to the young. The availability of precise measurements of BMD now facilitates the assessment of patients, the need for therapeutic intervention, and assessment of the response to therapy. The increasing number of approved, safe and effective therapeutic agents and the continuing intense research efforts bode well for improved skeletal health for women of all ages.

Risedronate in the treatment of Paget's disease of bone: an open label, multicenter study.

An open-label, multicenter study was conducted to determine the efficacy and safety of oral risedronate (a pyridinyl bisphosphonate) in 162 patients (102 men, 60 postmenopausal women; mean age, 68 years) with moderate to severe Paget's disease of bone (mean serum alkaline phosphatase [ALP] approximately seven times the upper limit of normal). Patients were treated with oral risedronate, 30 mg/day for 84 days, followed by 112 days without treatment. This 196-day cycle was repeated once if serum ALP did not normalize or increased from the nadir value by > or = 25%. At the end of the first and second cycles, the mean percentage decreases for serum ALP were 65.7% and 69.1%, and for urinary hydroxyproline/creatinine 50.4% and 66.9%, respectively. The decreases from baseline in ALP and urinary hydroxyproline/creatinine were significant (p < 0.001). Normalization of serum ALP was observed in 86 patients (53.8%): 53 during the first treatment cycle and 33 during the second. There was a significant proportion of patients reporting a decrease in the pagetic bone pain at days 84 and 196 (p < 0.001). Overall, risedronate was well tolerated. Five patients withdrew due to adverse events, none of which were considered to be drug related. In conclusion, 30 mg of oral risedronate administered daily for 84 days significantly reduced the biochemical indices of disease activity and was associated with pain reduction in patients with moderate to severe Paget's disease of bone. Normalization of ALP was observed in the majority of patients. Repeated administration of risedronate was shown to be beneficial. In general, risedronate was well tolerated and demonstrated a good safety profile.

Expression of parathyroid hormone-related protein (PTHrP) and the PTH/PTHrP receptor in the rat uterus during early pregnancy and following artificial deciduoma induction.

The interaction between parathyroid hormone-related protein (PTHrP) and the parathyroid hormone (PTH)/PTHrP receptor is thought to play a role in the growth and differentiation of various tissues throughout fetal development in the rodent. The aim of the present study was to define the patterns of expression of PTHrP and of the PTH/PTHrP receptor in the rat uterus during the early stages of normal pregnancy, and following artificial induction of a decidual reaction. Using hybridization histochemistry, we have shown that the receptor gene is switched on early in pregnancy (by 1.5 days post coitum) in the endometrial stromal cells that surround the lumen. These cells include the anti-mesometrial subepithelial stromal cells that are destined to become decidualized. This pattern continues until 5.0 days post coitum, when PTHrP is switched on in antimesometrial luminal epithelial cells that line the implantation chamber. Stromal cells underlying the implantation chamber then downregulate transcription of the receptor gene, and within 12 h differentiate into decidual cells. A similar pattern was seen in uteri in which a decidual reaction had been induced artificially. Therefore, it may be postulated that in early pregnancy the endometrial stroma initiates transcription of the gene for the PTH/PTHrP receptor and is thus 'primed' for the PTHrP signal from the luminal epithelial cells. Some time after receiving the signal, the endometrial stromal cells downregulate the receptor gene, and this appears to be a trigger for the terminal differentiation of the stromal cells into decidual cells. These results suggest that PTHrP, acting through the PTH/PTHrP receptor, plays a role in the initiation of a decidual reaction during early pregnancy by regulating the differentiation of endometrial stromal cells into decidual cells.

In vitro expansion of CD13+CD33+ dendritic cell precursors from multipotent progenitors is regulated by a discrete fas-mediated apoptotic schedule.

We provide new information on how apoptosis regulates the expansion and survival of dendritic cell (DC) elements during in vitro hematopoiesis. Functionally distinct apoptotic schedules were associated with different phases of DC development when multipotent CD34+ progenitor cells were treated with GM-CSF + TNF +/- SCF (c-kit ligand). During early phases of growth, unselected progenitors underwent apoptosis. During intermediate stages, high levels of apoptosis resulted in the preferential selection of DC precursors, as revealed by the massive expansion of DR+CD33+CD13+ cells. Late apoptosis was associated with the death of mature DCs. Apoptotic events surrounding the earlier periods were related to the exogenous addition of TNF-alpha and appeared to be mediated by fas. In contrast, those events associated with terminally differentiated DCs were fas independent because there was no correlation between fas expression and cell death. The bcl-2 protein family appeared to confer resistance to apoptotic death, as revealed by the high levels of bcl-2 and bclxL during peak DC development and in long-term DC cultures. We demonstrate that activation of distinct apoptotic programs regulates DC development and homeostasis. Although suppression of apoptosis may prolong the survival of late DC elements, an earlier apoptotic schedule appears to be required for the selective expansion of DC elements from multipotent progenitors. Our data also provides insight into the mechanism(s) of myeloid lineage selection by cytokines such as TNF-alpha, which may promote both cell death and survival.

Intravenously administered pamidronate for treating refractory Paget's disease of bone.

OBJECTIVE: To report our experience with the use of intravenously administered pamidronate in 16 patients with refractory Paget's disease of bone. METHODS: We describe our treatment regimen and outline serum alkaline phosphatase levels at baseline and after pamidronate therapy in our study cohort. In addition, we summarize clinical symptoms and response to treatment. RESULTS: Although clinical experience with pamidronate in the treatment of Paget's disease of bone has been limited in the United States, elsewhere it has been shown to be an effective agent that inhibits the increased osteoclastic activity characteristic of this disease. Accordingly, in 16 patients with Paget's disease unresponsive to various other therapies, we administered pamidronate intravenously at a dose of 30 mg in 500 mL of 5% dextrose in water during a 4-hour period once weekly for 6 weeks. Serum alkaline phosphatase was determined at baseline and at regular intervals for at least 12 months after the onset of therapy. Many of these patients reported relief of pain and an increased flexibility or range of motion after treatment. The only adverse effect reported by these patients was an acute-phase reaction during the first infusion in two patients. In all patients, serum alkaline phosphatase levels declined, and significant (P = 0.0012) decreases from baseline were noted within 6 weeks after the initial infusion. Maximal responses were generally seen within 6 months after treatment, serum alkaline phosphatase levels decreasing as much as 91% from baseline. CONCLUSION: These data suggest that intravenous pamidronate therapy is an effective alternative to calcitonin and etidronate in the treatment of Paget's disease of bone, particularly in those patients refractory to such agents.

Multilocus linkage of familial hyperkalaemia and hypertension, pseudohypoaldosteronism type II, to chromosomes 1q31-42 and 17p11-q21.

Essential hypertension is a common multifactorial trait. The molecular basis of a number of rare diseases that after blood pressure in humans has been established, identifying pathways that may be involved in more common forms of hypertension. Pseudohypoaldosteronism type II (PHAII, also known as familial hyperkalaemia and hypertension or Gordon's syndrome; OMIM #145260), is characterized by hyperkalaemia despite normal renal glomerular filtration, hypertension and correction of physiologic abnormalities by thiazide diuretics. Mild hyperchloremia, metabolic acidosis and suppressed plasma renin activity are variable associated findings. The pathogenesis of PHAII is unknown, although clinical studies indicate an abnormality in renal ion transport. As thiazide diuretics are among the most efficacious agents in the treatment of essential hypertension, understanding the pathogenesis of PHAII may be of relevance to more common forms of hypertension. Analysis of linkage in eight PHAII families showing autosomal dominant transmission demonstrates locus heterogeneity of this trait, with a multilocus lod score of 8.1 for linkage of PHAII to chromosomes 1q31-q42 and 17p11-q21. Interestingly, the chromosome-17 locus overlaps a syntenic interval in rat that contains a blood pressure quantitative trait locus (QTL). Our findings provide a first step toward identification of the molecular basis of PHAII.