Veliparib with temozolomide or carboplatin/paclitaxel versus placebo with carboplatin/paclitaxel in patients with BRCA1/2 locally recurrent/metastatic breast cancer: randomized phase II study.

Background: Homologous recombination defects in BRCA1/2-mutated tumors result in sensitivity to poly(ADP-ribose) polymerase inhibitors, which interfere with DNA damage repair. Veliparib, a potent poly(ADP-ribose) polymerase inhibitor, enhanced the antitumor activity of platinum agents and temozolomide in early phase clinical trials. This phase II study examined the safety and efficacy of intermittent veliparib with carboplatin/paclitaxel (VCP) or temozolomide (VT) in patients with BRCA1/2-mutated breast cancer. Patients and methods: Eligible patients ≥18 years with locally recurrent or metastatic breast cancer and a deleterious BRCA1/2 germline mutation were randomized 1 : 1 : 1 to VCP, VT, or placebo plus carboplatin/paclitaxel (PCP). Primary end point was progression-free survival (PFS); secondary end points included overall survival (OS) and overall response rate (ORR). Results: Of 290 randomized patients, 284 were BRCA+, confirmed by central laboratory. For VCP versus PCP, median PFS was 14.1 and 12.3 months, respectively [hazard ratio (HR) 0.789; 95% CI 0.536-1.162; P = 0.227], interim median OS 28.3 and 25.9 months (HR 0.750; 95% CI 0.503-1.117; P = 0.156), and ORR 77.8% and 61.3% (P = 0.027). For VT (versus PCP), median PFS was 7.4 months (HR 1.858; 95% CI 1.278-2.702; P = 0.001), interim median OS 19.1 months (HR 1.483; 95% CI 1.032-2.131; P = 0.032), and ORR 28.6% (P < 0.001). Safety profile was comparable between carboplatin/paclitaxel arms. Adverse events (all grades) of neutropenia, anemia, alopecia, and neuropathy were less frequent with VT versus PCP. Conclusion: Numerical but not statistically significant increases in both PFS and OS were observed in patients with BRCA1/2-mutated recurrent/metastatic breast cancer receiving VCP compared with PCP. The addition of veliparib to carboplatin/paclitaxel significantly improved ORR. There was no clinically meaningful increase in toxicity with VCP versus PCP. VT was inferior to PCP. An ongoing phase III trial is evaluating VCP versus PCP, with optional continuation single-agent therapy with veliparib/placebo if chemotherapy is discontinued without progression, in this patient population. Clinical trial information: NCT01506609.

Chromaffin-adrenocortical cell interactions: effects of chromaffin cell activation in adrenal cell cocultures.

Although the adrenal cortex and medulla are both involved in the maintenance of homeostasis and stress response, the functional importance of intra-adrenal interactions remains unclear. When primary cocultures of frog (Rana pipiens) adrenocortical and chromaffin cells were used, selective chromaffin cell activation dramatically affected both chromaffin and adrenocortical cells. Depolarization with 50 microm veratridine enhanced chromaffin cell neuronal phenotype, contacts with adrenocortical cells, and secretion of norepinephrine, epinephrine, and serotonin. Time-lapse video microscopy recorded the rapid establishment of growth cones on the activated chromaffin cell neurites, neurite branching, and outgrowth toward adrenocortical cells. Simultaneously, adrenocortical cells migrated toward chromaffin cells. Following chromaffin cell activation, adrenocortical cell Fos protein expression and corticosteroid secretion were increased, indicating that chromaffin cell modulation of adrenocortical cells is at the transcriptional level. These results provide evidence that intra-adrenal interactions affect cellular differentiation and modulate steroidogenesis. Furthermore, this suggests that the activity-related plasticity of chromaffin and adrenocortical cells is developmentally and physiologically important.

Frog chromaffin and adrenocortical cell co-cultures: a model for the study of medullary control of corticosteroidogenesis.

Phylogenetic, physiological and morphological evidence indicates that interactions between chromaffin and adrenocortical cells are involved in the differentiation and maintenance of function of both cell types. Chromaffin-adrenocortical interaction has become recognized as an important component of adrenocortical regulation; however, the mechanisms by which chromaffin cells modulate adrenocortical function are not well understood. To study directly chromaffin-adrenocortical cellular interactions, we developed primary frog (Rana pipiens) adrenal co-cultures. In these co-cultures, chromaffin cells extend processes that project towards or onto adrenocortical cells, mimicking their organization in vivo and indicating a potential for interaction between the two cell types. Cell survival and differentiation were optimized using a combination of NGF, FGF and histamine to enhance neurite outgrowth and fetal calf serum plus 10(-10) M ACTH to maintain steroidogenesis. Characterization of the cells by immunocytochemistry and histochemistry showed that chromaffin cells maintain expression of catecholamine biosynthetic enzymes and that adrenocortical cells maintain expression of steroidogenic enzymes. Furthermore, chromaffin cells release catecholamines upon stimulation with carbamylcholine or potassium while adrenocortical cells sustain a basal secretion rate of aldosterone and corticosterone that is augmented 10-40-fold by 0.1 nM to 10 nM ACTH. We therefore propose that these co-cultures serve as a useful model system to study the cellular and molecular mechanisms by which chromaffin cells modulate adrenocortical cell function.

Chromaffin cell depolarization results in modulation of corticosteroidogenesis in co-culture.

Previous morphological and physiological evidence indicates that the adrenal medulla can modulate adrenocortical steroidogenesis, most likely via paracrine or neuronal interactions. To study directly chromaffin-adrenocortical cellular interactions, we previously developed co-cultures of frog (Rana pipiens) adrenal (interrenal) cells. Importantly, chromaffin cells in these co-cultures extend processes that project toward or onto adrenocortical cells, thereby providing the substrate for direct autonomic regulation of adrenocortical function and also mimicking the organization in vivo. To test whether chromaffin cells in our co-cultures affect adrenocortical steroidogenesis, we used veratridine, a sodium ionophore, to depolarize chromaffin cells. Chronic veratridine (50 microM) results in increased corticosterone secretion on days 3 (950%), and 4 (350%). These results indicate that chromaffin cell activation results in the modulation of corticosteroidogenesis.

Viraemic transmission of Crimean-Congo haemorrhagic fever virus to ticks.

In order to determine the way in which vertebrates infected with Crimean-Congo haemorrhagic fever (CCHF) virus and potential ixodid tick vectors interact in nature, immature and adult ticks of several species were fed on viraemic mammals and then assayed for virus content at varying times after feeding. CCHF virus was not isolated from ticks of six species tested after feeding as adults and immature forms on sheep with viraemia of 10(2.5-3.2) LD 50/ml, nor from larval ticks fed on guinea-pigs and white-tailed rats with viraemia of 10(1.9-2.7) LD 50/ml. In contrast, virus was isolated from 10 of 152 pools of engorged adult ticks of 5 species that fed on cattle with viraemia of 10(1.5-2.7) LD 50/ml and from 3 of 137 female ticks after oviposition. Infection was transmitted to larval and nymphal Hyalomma truncatum and H. marginatum rufipes, but not to Rhipicephalus evertsi evertsi, from a scrub hare with viraemia of 10(4.2) LD 50/ml but only nymphal H. truncatum and H. m. rufipes became infected from scrub hares with viraemia of 10(2.6-2.7) LD 50/ml. Infection was transmitted trans-stadially in H. m. rufipes and H. truncatum infected as nymphae, and adult H. m. rufipes transmitted infection to a sheep. No evidence of transovarial transmission was found in larval progeny of ticks exposed to CCHF virus as adults on sheep and cattle or as immatures on scrub hares.

Field and laboratory investigation of Crimean-Congo haemorrhagic fever virus (Nairovirus, family Bunyaviridae) infection in birds.

In November 1984 a case of Crimean-Congo haemorrhagic fever (CCHF) occurred in a worker who became ill after slaughtering ostriches (Struthio camelus) on a farm near Oudtshoorn in the Cape province of South Africa. The diagnosis was confirmed by isolation of CCHF virus from the patient's serum and by demonstration of a specific antibody response. It was suspected that infection was acquired either by contact with ostrich blood or by inadvertently crushing infected Hyalomma ticks while skinning ostriches. Reversed passive haemagglutination-inhibition antibody to CCHF virus was detected in the sera of 22/92 ostriches from farms in Oudtshoorn district, including 6/9 from the farm where the patient worked, but not in the sera of 460 birds of 37 other species. In pathogenicity studies domestic chickens proved refractory to CCHF infection, but viraemia of low intensity (maximum titre 2.5 log10 mouse ic LD50/ml) followed by a transient antibody response occurred in blue-helmeted guinea fowl (Numidia meleagris). These results offer the first direct evidence that some bird species are susceptible to CCHF virus infection.

Epidemiologic and clinical features of Crimean-Congo hemorrhagic fever in southern Africa.

Following the diagnosis in 1981 of the first case of Crimean-Congo hemorrhagic fever (CCHF) in South Africa, an antibody survey was undertaken on cattle sera to determine the distribution of the virus and specific diagnostic tests were routinely applied to specimens from suspected cases of hemorrhagic fever to establish the medical significance of its presence. Antibody to CCHF virus was demonstrated by reversed passive hemagglutination-inhibition technique in 2,460/8,667 (28%) cattle sera and in 140/180 herds tested in South Africa, as well as in 347/763 (45%) cattle sera and in 32/34 (94%) herds tested in Zimbabwe. The antibody was found in all major cattle farming areas, but was of low prevalence along the southern coast where 2 of the 3 species of Hyalomma tick which occur in South Africa are absent. From February 1981 to January 1986, inclusive, 29 indigenous cases of CCHF were diagnosed in 16 outbreaks which arose in various locations throughout South Africa. A further 2 imported cases of CCHF arose in Zaire and Tanzania. The clinical features of infection conformed to the classical descriptions of CCHF in the Soviet Union. The fatal outcome in 11/31 cases indicates that the African disease is no less severe than that which occurs in Eurasia. It is inferred that the virus is widespread in all countries in Africa and Eurasia which lie within the limits of world distribution of ticks of the genus Hyalomma.

Antibody to Crimean-Congo hemorrhagic fever virus in wild mammals from southern Africa.

Crimean-Congo hemorrhagic fever (CCHF) virus is becoming increasingly recognized as an important human pathogen in southern Africa. In order to determine the role of wild mammals in the natural ecology of the virus, sera from 3,772 wild mammals of 87 species and from 1,978 domestic dogs collected in South Africa and Zimbabwe between 1964 and 1985 were tested for antibody to CCHF virus by reversed passive hemagglutination inhibition (RPHI) and by indirect immunofluorescence (IF). Antibody was found to be highly prevalent in large mammals in the Orders Artiodactyla and Perissodactyla such as giraffe, Giraffa camelopardalis (3/3 positive), rhinoceros, Ceratotherium simium and Diceros bicornis (7/13), eland, Taurotragus oryx (59/127), buffalo, Syncerus caffer (56/287), kudu, Tragelaphus strepsiceros (17/78), and zebra, Equus burchelli (16/93). In small mammals antibody was found in the sera of 40/293 hares, 22/1,305 rodents, and 1/74 wild carnivores, but not in 522 primates, 176 insectivores, or 19 hyrax. Antibody was also found in the sera of 118/1,978 domestic dogs. The species of wild mammal in which antibody was distributed (with highest antibody prevalence in hares and large herbivores) reflects the feeding preference of immature and adult ticks of the genus Hyalomma, suggesting that Hyalomma sp. are the principal CCHF vectors in the wild.

Comparison of techniques for demonstrating antibodies to Rift Valley fever virus.

Nine serological techniques were compared by monitoring the response to infection with Rift Valley fever (RVF) virus in three sheep. Antibodies were monitored daily for the first 14 days after infection, then weekly and later fortnightly up to week 24. The earliest antibody response was detected in one sheep on day 3 by a plaque reduction neutralization test, and by day 6 antibodies were demonstrable in all three sheep by haemagglutination-inhibition, reversed passive haemagglutination-inhibition, immunodiffusion, indirect immunofluorescence (IF), enzyme-linked immunosorbent assay and neutralization of cytopathic effect in cell cultures. Antibodies were demonstrable by complement fixation on day 8 at the earliest. IF and the two neutralization techniques produced the highest titres, but all tests could be used satisfactorily for the serological diagnosis of RVF. Inactivated antigen could be used for all except the neutralization tests. A radioimmunoassay technique using 125I-labelled staphylococcal protein A detected antibodies on day 8 at the earliest and produced lower mean titres than some of the other techniques. This was probably because sheep immunoglobulins bind protein A poorly.

Comparative pathogenicity and antigenic cross-reactivity of Rift Valley fever and other African phleboviruses in sheep.

Homologous and heterologous haemagglutination-inhibition (HAI), complement-fixation (CF), immunodiffusion (ID) and mouse neutralization tests were performed with the Lunyo (LUN) and a Zimbabwean strain of Rift Valley fever (RVF) virus, the prototype and a South African strain of Arumowot (AMT) virus and prototype strains of Gordil (GOR), Saint-Floris (SAF) and Gabek Forest (GF) viruses, using immune mouse ascitic fluids prepared against these viruses. Reactions of identity occurred in all tests between LUN and the Zimbabwean strains of RVF and between the two strains of AMT virus. Otherwise, cross-reactions occurred between all the phleboviruses in HAI tests, while reactions in CF, ID and neutralization tests were monospecific for virus serotypes, except that weak cross-reaction occurred between GOR and SAF viruses in CF and ID tests. Four sheep infected subcutaneously with the Zimbabwean strain of RVF virus developed transient fever, viraemia, leucopaenia, relative thrombocytopaenia, haemoconcentration and raised serum enzyme levels, which indicated that the sheep had developed necrotic hepatitis. Disseminated focal necrotic hepatitis was confirmed in a sheep killed for examination on day 4 post-infection. The other three sheep recovered uneventfully after only mild depression and anorexia. Groups of three sheep infected with SAF, GOR, AMT and GF viruses had no demonstrable viraemia or other sign of infection or illness, except that the sheep infected with AMT developed mild fever lasting less than 24 h. Antibody responses were monitored at intervals over a period of 24 weeks in all sheep by homologous and heterologous HAI, CF and cell culture neutralization (CPENT) tests. Homologous antibody responses were marked in the RVF-infected sheep and their sera cross-reacted strongly in HAI tests with antigens of the other viruses. The sera of the RVF-infected sheep cross-reacted less markedly in CF and CPENT tests. Homologous antibody responses were poor in all the sheep infected with phleboviruses other than RVF, and the cross-reactivity of their sera for RVF antigen or virus was negligible. All sheep were challenged with RVF virus 48 weeks after their initial infection. The sheep which had originally been infected with RVF virus were immune and developed neither fever nor viraemia. All other sheep developed fever, viraemia and antibodies to RVF virus. It was concluded that the African phleboviruses, other than RVF, are unlikely to cause disease in livestock or to induce antibodies which could cause confusion in the diagnosis of RVF.

Comparison of methods for isolation and titration of Crimean-Congo hemorrhagic fever virus.

The fluorescence focus assay and the plaque assay in CER cells were compared with mouse inoculation for the isolation and titration of Crimean-Congo hemorrhagic fever virus. The fluorescence focus assay and the plaque assay were of similar sensitivity, but both produced 10- to 100-fold lower titers than did mouse inoculation. For specimens from 26 Crimean-Congo hemorrhagic fever patients in South Africa, virus was isolated from 20 by mouse inoculation and from only 11 by cell culturing. Although cell cultures were less sensitive for the isolation of virus from clinical specimens, they produced diagnostic results much more rapidly.

A nosocomial outbreak of Crimean-Congo haemorrhagic fever at Tygerberg Hospital. Part V. Virological and serological observations.

A nosocomial outbreak of Crimean-Congo haemorrhagic fever occurred in Tygerberg Hospital near Cape Town in September 1984 when 7 medical personnel became ill after admission of an index patient. The disease was fatal in the index and 1 secondary case, and was confirmed in the index and 6 secondary cases by isolation of the virus. An antibody response was demonstrated in the remaining patient, thought to be a tertiary case, but the fact that the patient received immune plasma therapy raises doubts about the validity of the diagnosis. The index case had contact with ticks and horses but his infection could not be related to a specific incident.

A common-source outbreak of Crimean-Congo haemorrhagic fever on a dairy farm.

An outbreak of Crimean-Congo haemorrhagic fever (CCHF) on a dairy farm in the Orange Free State in 1984 is described. Forty-six cows were purchased from the western Cape Province in January 1984; 2 died from the tick-borne disease anaplasmosis in March and a labourer who helped butcher the carcasses became ill a few days later. Another cow died at the end of April and within 9 days 4 people who had come into contact with its blood became ill. Antibodies to CCHF virus were found in the sera of the 5 patients but not in other residents of the farm. Three patients recovered from a severe influenza-like illness without seeking medical attention; 1 patient, who was admitted to hospital, recovered from illness marked by haematemesis, epistaxis and amnesia and the 5th patient died of complications of surgery for brain haemorrhage. Antibody studies indicated that many of the cows became infected with CCHF after their arrival on the farm. It can be deduced that animals reared in tick-free, or relatively tick-free, circumstances, which are then moved to where they are subject to heavy parasitization by ticks, can acquire common tick-borne diseases of livestock plus CCHF infection simultaneously. In such circumstances there is a definite risk of human exposure to CCHF-infected blood or other tissues.

Investigations following initial recognition of Crimean-Congo haemorrhagic fever in South Africa and the diagnosis of 2 further cases.

Sera from 124 cattle herds were tested, and antibodies to Crimean-Congo haemorrhagic fever (CCHF) were found in 93 herds. The prevalence of antibodies was high in the interior of the country, in excess of 90% in some herds, but was less than 4% in cattle along the coast from Cape Town to East London. Only 17 out of 1109 (1,5%) human residents of 55 farms had antibodies to CCHF, while none of 164 veterinary research workers or 98 veterinarians engaged in farm animal practice had them. Specimens from 130 suspected cases of viral haemorrhagic fever were examined and CCHF was diagnosed only in the patient previously reported as the first case of the disease to be recognized in this country. A further 2 cases of CCHF were diagnosed by examining 318 specimens from patients with nonfatal febrile illness. Both patients had contact with livestock. Increasing awareness of the disease will probably lead to an increase in the number of cases diagnosed, but there are no grounds for concluding that the disease is on the increase.

Protein synthesis in Rift Valley fever virus-infected cells.

Rift Valley fever virus-induced protein synthesis was examined by polyacrylamide gel electrophoresis and fluorography. Five virus-induced polypeptides were detected, the nucleocapsid protein N, the nucleus-associated nonstructural protein NS1, the glycoproteins G1 and G2, and a protein of molecular weight 80K. The N, G1, G2, and 80K proteins were present in virion preparations. Sequential studies showed that NS1 accumulated in the nucleus as soon as it was formed and readily associated with nuclei partitioned from noninfected cells. The G1 and G2 proteins labelled with [3H]glucosamine and [3H]mannose. NS1 was shown to be the only virus-induced protein which was phosphorylated.