Advances in the treatment of cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) is a threat to immunologically weak patients. HCMV cannot yet be eliminated with a vaccine, despite recent advances. SOURCES OF DATA: Sources of data are recently published research papers and reviews about HCMV treatments. AREAS OF AGREEMENT: Current antivirals target the UL54 DNA polymerase and are limited by nephrotoxicity and viral resistance. Promisingly, letermovir targets the HCMV terminase complex and has been recently approved by the FDA and EMA. AREAS OF CONTROVERSY: Should we screen newborns for HCMV, and use antivirals to treat sensorineural hearing loss after congenital HCMV infection? GROWING POINTS: Growing points are developing drugs against latently infected cells. In addition to small molecule inhibitors, a chemokine-based fusion toxin protein, F49A-FTP, has shown promise in killing both lytically and latently infected cells. AREAS TIMELY FOR DEVELOPING RESEARCH: We need to understand what immune responses are required to control HCMV, and how best to raise these immune responses with a vaccine.

Diverse specificities, phenotypes, and antiviral activities of cytomegalovirus-specific CD8+ T cells.

UNLABELLED: CD8(+) T cells specific for pp65, IE1, and IE2 are present at high frequencies in human cytomegalovirus (HCMV)-seropositive individuals, and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described, but little is known about their phenotypes and functions. Consequently, in this study, we chose to determine the diversity of HCMV-specific CD8(+) T cell responses to the products of 11 HCMV open reading frames (ORFs) in a cohort of donors aged 20 to 80 years old as well as the ability of the T cells to secrete gamma interferon (IFN-γ). Finally, we also tested their functional antiviral capacity using a novel viral dissemination assay. We identified substantial CD8(+) T cell responses by IFN-γ enzyme-linked immunospot (ELISPOT) assays to all 11 of these HCMV proteins, and across the cohort, individuals displayed a range of responses, from tightly focused to highly diverse, which were stable over time. CD8(+) T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA(+), CD57(+), and CD28(-), across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex vivo isolation. Taken together, our data argue that HCMV-specific CD8(+) T cells have effective antiviral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion. IMPORTANCE: Human cytomegalovirus (HCMV) is normally carried without clinical symptoms and is widely prevalent in the population; however, it often causes severe clinical disease in individuals with compromised immune responses. HCMV is never cleared after primary infection but persists in the host for life. In HCMV carriers, the immune response to HCMV includes large numbers of virus-specific immune cells, and the virus has evolved many mechanisms to evade the immune response. While this immune response seems to protect healthy people from subsequent disease, the virus is never eliminated. It has been suggested that this continuous surveillance by the immune system may have deleterious effects in later life. The study presented in this paper examined immune responses from a cohort of donors and shows that these immune cells are effective at controlling the virus and can overcome the virus' lytic cycle immune evasion mechanisms.

LPS promotes a monocyte phenotype permissive for human cytomegalovirus immediate-early gene expression upon infection but not reactivation from latency.

Human cytomegalovirus (HCMV) infection of myeloid cells is closely linked with the differentiation status of the cell. Haematopoietic progenitors and CD14+ monocytes are usually non-permissive for lytic gene expression which can lead to the establishment of latent infections. In contrast, differentiation to macrophage or dendritic cell (DC) phenotypes promotes viral reactivation or renders them permissive for lytic infection. The observation that high doses of Lipopolysaccharide (LPS) drove rapid monocyte differentiation in mice led us to investigate the response of human monocytes to HCMV following LPS stimulation in vitro. Here we report that LPS triggers a monocyte phenotype permissiveness for lytic infection directly correlating with LPS concentration. In contrast, addition of LPS directly to latently infected monocytes was not sufficient to trigger viral reactivation which is likely linked with the failure of the monocytes to differentiate to a DC phenotype. Interestingly, we observe that this effect on lytic infection of monocytes is transient, appears to be dependent on COX-2 activation and does not result in a full productive infection. Thus LPS stimulated monocytes are partially permissive lytic gene expression but did not have long term impact on monocyte identity regarding their differentiation and susceptibility for the full lytic cycle of HCMV.

Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

Hypercalcemia associated with chronic lymphocytic leukemia.

A patient with chronic lymphocytic leukemia (CLL) is described in whom hypercalcemia occurred in association with elevation of the peripheral lymphocyte count and expansion of total tumor mass. Hypercalcemia was ameliorated with the institution of chemotherapy for the leukemic process and subsequent fall in WBC count and decrease in total tumor burden; hypercalcemia recurred with relapse of the leukemic process. The serum immunoreactive parathyroid hormone (iPTH) concentration, when measured, was inappropriately elevated for the degree of hypercalcemia. The hypercalcemia would appear to be a direct consequence of the leukemia, and possibly involved secretion of a parathyroid hormone-like polypeptide by the CLL cells. Although a possible role for either an osteoclast-activating substance or prostaglandins was not excluded, they would not account for the elevated serum iPTH levels observed.

Physiological properties of the luteinizing hormone pulse signal: impact of intensive and extended venous sampling paradigms on its characterization in healthy men and women.

The pulsatile nature of the gonadotropin signal is a critical determinant of physiological activation of the gonadal axis. Nonetheless, major uncertainties exist regarding the exact patterns of LH secretion that constitute normal physiological profiles in man. To assess possible bases for the discrepancies in the literature, we sampled blood at 5-min intervals for 24 h in eight normal men and eight normal women in the early follicular phase of the menstrual cycle. The constituent 5-, 10-, 15-, and 20-min immunoactive LH series and the consituent 6-, 12-, or 24-h sampling durations provided ranges of sampling intensities and durations for analysis of significant LH pulses. A technique for minimizing the influence of false positive immunoassay errors on peak detection was used to aid in estimating apparent true positive LH pulse frequency. Nonlinear curve fitting of the relationship between sampling intensity and apparent true positive LH pulse frequency revealed a stable pulse frequency estimate at intensive rates of venous sampling, with values of 19.5 +/- 1.9 (+/- SEM) pulses/24 h (periodicity, 73.8 +/- 6.5 min/pulse) in men and 20.6 +/- 3.6 pulses/24 h (periodicity, 70.0 +/- 10 min/pulse) in women. Further analyses indicated that sampling every 3.1 and 2.0 min for 24 h would be required to capture 90% of the LH pulses in men and women, respectively. Moreover, even with a 5-min sampling rate, the statistical counting errors of the LH pulse frequency estimates varied markedly with sampling duration; for example, in men sampled in 6-, 12-, and 24-hr sessions, the values were, respectively, 49%, 35%, and 24% of the observed pulse frequencies. Similar variations were found in women. Counting errors were 30-50% higher for conventional 20-min sampling rates than for 5-min sampling intervals. Measured interpulse intervals varied widely from 10-330 min (median, 60 min; n = 131 LH pulses) in men and from 10-340 min (median, 65 min; n = 125 LH pulses) in women. In addition, absolute LH pulse amplitudes varied from 1-28 mIU/ml (median, 4.1 mIU/ml) in men and from 1-24 mIU/ml (median, 3.6 mIU/ml) in women. These estimates were associated with a median number of points identified within each pulse of 6.0 in men and 4.0 in women. In summary, the present exhaustive sampling studies demonstrate for the first time stable estimates of physiological LH pulsations in normal men and women, and document broad ranges of normal LH pulse amplitudes and interpulse intervals.(ABSTRACT TRUNCATED AT 400 WORDS)

Rickets in adult cystic fibrosis with myopathy, pancreatic insufficiency and proximal renal tubular dysfunction.

Rickets is reported in a 19 year old white man with cystic fibrosis in whom pancreatic and hepatic involvement was advanced. There was evidence of secondary hyperparathyroidism with proximal renal tubular acidosis, aminoaciduria, phosphaturia and hypophosphatemia. Treatment with oral pancreatic and parenteral vitamin D supplements led to full recovery of the rachitic syndrome and the proximal renal tubular dysfunction.

Phenytoin inhibition of parathyroid hormone induced bone resorption in vitro.

1 A study is reported of the effects of phenytoin and phenobarbitone on bone calcium mobilization by parathyroid hormone in vitro.2 In a therapeutic concentration (15 mug/ml), phenytoin significantly inhibited parathyroid hormone-induced calcium release from bone.3 The inhibitory effect of phenytoin on bone calcium mobilization could play a role in the maintenance of hypocalcaemia in epileptic patients on long-term anticonvulsant drug therapy.

Magnesium and potassium. Inter-relationships in cardiac disorders.

Magnesium and potassium are the 2 major intracellular cations. The intracellular concentrations of these 2 ions appear to be closely correlated, but the existence of a relationship between the plasma concentrations of these ions has been controversial. A major function of potassium is to maintain the excitability of nerve and muscle tissue. Alterations in either the intracellular or extracellular potassium concentration affect membrane excitability by alterations in the resting membrane potential. The critical factor is not the actual potassium concentration in either compartment but rather the ratio of the intracellular to the extracellular concentration. The intracellular concentration of potassium is maintained against an electrochemical gradient by active transport involving an ionic pump mechanism. In this metabolically active ion-pump mechanism the inward transport of potassium is balanced by the outward transport of sodium; this active transport mechanism involves magnesium. The plasma concentration of potassium has been reported to be an important factor in the genesis of cardiac arrhythmias. The arrhythmogenic mechanisms of hypokalaemia, particularly in mild degrees, have not been clearly defined. However, evidence has begun to accumulate that magnesium deficiency may be a critical factor in the cardiac arrhythmias associated with hypokalaemia. Diuretic drugs are recognised as primary agents in the treatment of essential hypertension. In patients on antihypertensive treatment evidence has recently been reported that there is a link between the administration of diuretics and sudden death. In addition to their action on the renal tubular handling of sodium and water, diuretic drugs affect the renal tubular handling of other ions. A well-established complication of therapy with diuretic drugs is an increased urinary excretion of potassium resulting in hypokalaemia. Hypokalaemia and hypomagnesaemia can be induced by the same mechanisms and are often clinically correlated with one another. The reported incidence of hypomagnesaemia is greater than that of hypokalaemia; a significant correlation also appears to exist between the plasma concentrations of magnesium and potassium. A significant inter-relationship between the plasma concentrations of magnesium and potassium and the evidence for a critical role of magnesium in the genesis of cardiac arrhythmias would support the proposal that magnesium should be routinely measured in situations, such as diuretic therapy, that are potentially associated with hypokalaemia.(ABSTRACT TRUNCATED AT 400 WORDS)

Water content of aluminum, dialysis dementia, and osteomalacia.

In the presence of normal renal function, a high concentration of aluminum in drinking water has been implicated as a factor in the etiology of a neurological syndrome in one specific geographical area. The role of aluminum as a toxic agent in other neurological disorders, where renal function is normal, is controversial. Aluminum is absorbed from the gastrointestinal tract and is normally excreted by the kidneys in the urine. In patients with chronic renal failure, aluminum appears to be of proven toxicological importance. In these patients the accumulation of aluminum in tissues causes an encephalopathy (dialysis encephalopathy or dialysis dementia), a specific form of metabolic bone disease (osteomalacic dialysis osteodystrophy), and an anemia and also plays an etiological role in some of the other complications associated with end-stage chronic renal disease. A failure in the normal renal excretory mechanism accounts for the tissue accumulation in chronic renal failure. The majority of chronic renal failure patients who develop aluminum toxicity are on long-term treatment with either hemo- or peritoneal dialysis; some patients develop toxicity who are only on treatment with aluminum-containing phosphate-binding agents. Aluminum in the dialysate appears to be the major source of the metal in chronic renal failure patients who develop aluminum toxicity. The aluminum content of the dialysate depends primarily on the content of the water with which it is prepared; there may be some contribution from the chemicals used in the concentrate which is added to the water. Some domestic tap-water supplies contain aluminum in high concentration, either naturally or because aluminum has been added as a flocculant in the purification process.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid centrifugal analyzer enzyme immunoassays for phenytoin, phenobarbital, and primidone.

The adaptation of the homogeneous enzyme immunoassays (EMIT) for phenytoin, phenobarbital, and primidone to a centrifugal analyzer is described. The sample volume required was 10 microliter, and the assay had the capacity to analyze sera from 28 patients within 180 sec. The assay temperature was 30 C, and absorbance was monitored at 340 nm. Coefficients of variation for within-day precision ranged from 2.1% to 3.7%, and analytic recovery was quantitative. The centrifugal analyzer EMIT assay results correlated well with those obtained using high-pressure liquid and gas-liquid chromatographic technics. A logit-log transformation of the absorbance rate versus concentration data was obtained using a modified Gauss-Newtonian nonlinear least-squares fit analysis. Severe hemolysis and lipemia caused interference.

A multivariate approach to prognostication in experimental bacterial meningitis.

Known concentrations of type III pneumococci were inoculated into eighty-one rabbits by cisternal puncture. Antibiotic therapy was started the following day. Aliquots of cerebrospinal fluid (CSF) and plasma were sampled on day one immediately before therapy was started and at regular intervals thereafter for up to eight days. Samples were analyzed for glucose, lactate, lactic dehydrogenase, and creatine kinase in various combinations. Leukocyte counts were performed on all CSF specimens. The timing of the specimens proved critical to the prognostic utility of the analyses performed. Day two plasma glucose was the most important single measurement for prognostication. Day one values for CSF glucose, lactate, and leukocyte count were also important. Substantial gains in prognostic accuracy were achieved when clinical laboratory measurements were used in combination by discriminant function analysis.

The urinary calcium-creatinine ratio as a measure of urinary calcium excretion.

Urine specimens were collected from 26 normal subjects, 10 patients with proven primary hyperparathyroidism, and eight patients with hypercalcaemia due to other causes. After overnight urine concentration, an oral water load was given to induce a diuresis and provide urine specimens with a relatively wide range of creatinine concentration for each subject. In normal subjects the urinary calcium/creatinine ratio was found to be independent of urine concentration. In eight out of 10 patients with primary hyperparathyroidism and in two out of eight patients with hyper-calcaemia due to other causes, the urinary calcium/creatinine ratio was found to be high when the creatinine concentration was low, but usually normal when the creatinine concentration was high. The results suggest that if the urinary calcium/creatinine ratio of random urine specimens is used as a ;screening' procedure to detect hypercalciuria the latter cannot be excluded if the urinary creatinine concentration is more than 40 mg per 100 ml.

The effect of diurnal variation on total plasma calcium concentration in normal subjects.

In 12 normal subjects plasma calcium and phosphorus concentrations were estimated during a normal working day. In the individual subjects fluctuations from the fasting values occurred during the period of study, but at no time during that period did the mean values differ significantly from the fasting plasma calcium or phosphorus concentrations. In three of the subjects the study was repeated while fasting during a normal working day. In these subjects the fluctuations in both plasma calcium and phosphorus concentrations were less than during a normal day while taking meals, and there was evidence of a diurnal variation in the plasma phosphorus concentration.

Value of plasma chloride concentration and acid-base status in the differential diagnosis of hyperparathyroidism from other causes of hypercalcaemia.

A study is reported of the estimation of plasma chloride concentration and acid-base status in the differentiation of primary hyperparathyroidism from all other causes of hypercalcaemia. In the two groups of patients studied, all of whom had hypercalcaemia, there was complete separation between the two groups on the basis of plasma chloride concentration and acid-base status. In 16 patients with primary hyperparathyroidism the increase in plasma chloride concentration and associated metabolic acidosis could have been accounted for by the known renal tubular effects of parathyroid hormone. In 13 patients with hypercalcaemia due to various other causes the decrease in plasma chloride concentration and associated metabolic alkalosis could be accounted for either by the known effects of an excess of calcium-ion on the renal tubules, or perhaps by suppression of endogenous parathyroid hormone secretion. In patients with hypercalcaemia and hypophosphataemia of ;pseudohyperparathyroidism' associated with non-endocrine tumours it is postulated that the low plasma chloride concentrations and metabolic alkalosis found in these patients were due either to a differing biological activity of the parathyroid-hormone-like polypeptide secreted by the tumour cells, or possibly to simultaneous secretion by these cells of an ACTH-like polypeptide.

Plasma "cortisol" levels in right and left ventricular failure.

Plasma ;cortisol' values are reported in 36 patients during or following right ventricular failure, and 12 during or following left ventricular failure. In patients with right ventricular failure the normal circadian rhythm was abolished with elevation of midnight values. Disturbance of rhythm was less marked in left ventricular failure, although midnight values were usually raised. Dexamethasone suppression of plasma ;cortisol' levels was abolished or reduced in right ventricular failure. These results confirm our earlier findings and further support our contention that the disturbance of circadian rhythm is not due to a simple stress mechanism. The results are of importance in the differential diagnosis of Cushing's syndrome based on plasma ;cortisol' values, if there is concomitant right ventricular failure.

Plasma calcium fractions and the protein-binding of calcium in normal subjects and in patients with hypercalcaemia and hypocalcaemia.

A study is reported of the estimation of plasma calcium fractions and the calcium-binding affinity of plasma proteins in a total sample of 59 people, which included 29 normal subjects and 30 patients with either hypercalcaemia or hypocalcaemia. It was demonstrated that when the sample was considered as a whole there was a significant correlation between the total plasma calcium concentration and the ultrafiltrable, ionized, and protein-bound calcium fractions and between the ultrafiltrable and ionized fractions. We have also demonstrated that in patients with either hypercalcaemia or hypocalcaemia, including acidotic uraemics, the calcium-binding affinity of the plasma proteins did not differ significantly from that in normal subjects. A significant correlation was also found between the total plasma calcium concentration and the ultrafiltrable, ionized and protein-bound calcium fractions when the normal subjects and the groups of patients with hypercalcaemia and hypocalcaemia due to chronic renal failure were considered as separate groups. The group of patients with hypercalcaemia included patients both with hyperparathyroidism and with hypercalcaemia due to other causes.

Ultramicrofluorimetric determination of calcium in plasma.

A precise and reproducible method is described for the ultramicro fluorimetric determination of calcium in plasma, based upon the formation of a fluorescent complex between calcium and calcein at a strongly alkaline pH. In a group of normal subjects, the mean concentration of plasma calcium was 9.66 mg/100 ml with a normal range of 8.9 to 10.4 mg/100 ml.

Quantitative image analysis of temporal changes in tau and neurofilament proteins during the course of acute experimental neurofibrillary degeneration; non-phosphorylated epitopes precede phosphorylation.

Perturbation of the neuronal cytoskeleton represents an integral feature of neurofibrillary tangles which are characteristic neuropathological findings seen in Alzheimer's disease. Microtubule associated protein tau (tau) is considered to be the major component of these lesions although neurofilament proteins also are present. The present study explores the formation of intraneuronal tau and neurofilament protein aggregates using intracisternal administration of aluminum maltolate to rabbits. The time course of the formation of these aggregates and subsequent phosphorylation have been investigated by immunohistochemical methods using a panel of monoclonal antibodies, with quantitation of the staining by image analysis. Neurofilament proteins begin to aggregate by day 1 following aluminum maltolate injection on day 0. Increases in non-phosphorylated neurofilament proteins are observed first, with phosphorylated epitopes being recognized by day 3. Tau follows a similar pattern in that non-phosphorylated epitopes appear to precede phosphorylation. The monoclonal antibody Alz-50 which recognizes a phosphorylation-independent epitope of tau in Alzheimer's disease paired helical filaments, demonstrates positivity in the aluminum maltolate-treated rabbits by day 3. Other tau monoclonal antibodies which recognize phosphorylated tau in paired helical filaments (AT8 and PHF-1) show positive immunostaining on days 6-8. These results indicate that intraneuronal aggregation of cytoskeletal proteins can be initiated by factors other than phosphorylation. However, phosphorylation occurring as a secondary event probably contributes to stabilization of the aggregates.

Tau immunoreactivity associated with aluminum maltolate-induced neurofibrillary degeneration in rabbits.

Intracisternal administration of aluminum maltolate to rabbits produces a marked argyrophilic neurofibrillary degeneration (NFD) which is also immunoreactive for both phosphorylated and non-phosphorylated microtubule associated protein tau. Using tissue fixation in PBF, the monoclonal antibodies Tau-2 and AT8 stain the NFD. Dephosphorylation markedly reduces the positivity of AT8. Using PLP-fixed tissue, monoclonal antibody Tau-1 also immunostains aluminum-induced NFD.