Calreticulin binding and other biological activities of survival peptide Y-P30 including effects of systemic treatment of rats.

Neuron survival-promoting peptide Y-P30, purified from oxidatively stressed neural cell lines, inhibits the appearance of microglia and rescues neurons 1 week after direct application to lesions of the rat cerebral cortex (7). Y-P30 affinity matrices treated with solubilized membranes from a variety of cell lines including human neuroblastoma SY5Y, mouse hippocampal cells HN 33.1, and human promonocytes HL-60, as well as with cerebral cortex tissue from both humans and rats, showed highly specific binding to calreticulin, a ubiquitous calcium binding protein that may be critical for integrin function. Treatment of cultures with 0.1 nM Y-P30 stabilized all these cell types whether differentiated or not, while 1 microM peptide also inhibited the morphological differentiation of the HL-60 cells into macrophages. Western analysis of the medium of SY5Y cell cultures suggested Y-P30-stimulated release of calreticulin, a result consistent with its other biological activities. Likewise, single dose systemic application of Y-P30 in unoperated rats and in rats with cerebral cortex lesions produced significant reductions in cerebral cortex membrane-associated calreticulin. Both direct and intravenous treatment with peptide also reduced cortical neuron atrophy 4 days after these lesions but only direct application consistently inhibited the appearance of ED-1(+) monocyte derivatives. We suggest that in vitro and in vivo mechanisms of Y-P30 effects are similar and involve the targeting of calreticulin. The results also suggest that some of these activities are apparent in the cerebral cortex after systemic application of this peptide.

Identification of a survival-promoting peptide in medium conditioned by oxidatively stressed cell lines of nervous system origin.

A survival-promoting peptide has been purified from medium conditioned by Y79 human retinoblastoma cells and a mouse hippocampal cell line (HN 33.1) exposed to H2O2. A 30 residue synthetic peptide was made on the basis of N-terminal sequences obtained during purification, and it was found to exhibit gel mobility and staining properties similar to the purified molecules. The peptide maintains cells and their processes in vitro for the HN 33.1 cell line treated with H2O2, and in vivo for cortical neurons after lesions of the cerebral cortex. It has weak homology with a fragment of a putative bacterial antigen and, like that molecule, binds IgG. The peptide also contains a motif reminiscent of a critical sequence in the catalytic region of calcineurin-type phosphatases; surprisingly, like several members of this family, the peptide catalyzes the hydrolysis of para-nitrophenylphosphate in the presence of Mn2+. Application of the peptide to one side of bilateral cerebral cortex lesions centered on area 2 in rats results in an increase in IgG immunoreactivity in the vicinity of the lesions 7 d after surgery. Microglia immunopositive for IgG and ED-1 are, however, dramatically reduced around the lesions in the treated hemisphere. Furthermore, pyramidal neurons that would normally shrink, die, or disintegrate were maintained, as determined by MAP2 immunocytochemistry and Nissl staining. These survival effects were often found in both hemispheres. The results suggest that this peptide operates by diffusion to regulate the immune response and thereby rescue neurons that would usually degenerate after cortical lesions. The phosphatase activity of this molecule also suggests the potential for direct neuron survival-promoting effects.

Induction of opioid receptor-mediated macrophage chemotactic activity after neonatal brain injury.

Macrophages have a prominent role in the injury response of the brain, yet the molecular mechanisms that control their invasion to the site of neuronal degeneration is unknown. After removal of the posterior cortex at birth, there is massive and specific targeting of nonresident macrophages to axotomized neurons in the lateral thalamus. The present study has identified an injury-induced, brain-derived chemotactic factor (BDCF) capable of eliciting chemotactic responses from resident peritoneal macrophages and brain macrophages. Conditioned media collected from tissue slices containing the axotomized central nervous system neurons exhibit BDCF activity. Initial experiments indicated that BDCF is a small peptide and, thus, we used specific pharmacologic reagents to characterize further BDCF activity. Naloxone, a pan opioid receptor antagonist, completely blocks BDCF activity. Although both kappa and mu opioid receptor antagonists failed to modify BDCF-induced macrophage chemotaxis, two specific delta receptor antagonists blocked BDCF. Analysis of BDCF by reverse phase HPLC and RIA revealed peak chemotactic activity in fractions consistent with the presence of an opioid peptide. The results suggest that cells in the brain respond to neuronal injury by producing and releasing opioids that can initiate a specific macrophage response.

Clinical triggers for detection of fever and dehydration. Implications for long-term care nursing.

1. Fever is a common problem among long-term care residents, and the clinical manifestations of fever and infections may be vague or nonspecific. 2. The majority of fevers in this study were staff-detected versus resident-initiated; this implies that staff vigilance is important in the detection of fever. 3. Staff documentation of impaired oral intake during febrile episodes was associated highly with either elevated serum sodium or blood urea nitrogen/creatinine ratios. Therefore, nursing assessment and interventions to hydrate residents at the first indication of impaired oral intake may prevent dehydration. 4. Routine mandated vital signs were found to be of little or no value in detecting fevers.

NMDA receptor blockade rescues Clarke's and red nucleus neurons after spinal hemisection.

Hemisection of the adult rat spinal cord at T9 transects the ascending ipsilateral axons of Clarke's nucleus (CN) neurons and the descending contralateral axons of red nucleus (RN) neurons. Eight weeks following axotomy, 30% of CN neurons and 22% of RN neurons die. Since both nuclei receive glutamatergic input, we wished to examine the possibility that glutamatergic excitotoxicity contributes to axotomy-induced neuronal death in these nuclei. To test this we studied the effects of administration of the NMDA receptor antagonist MK-801 on cell survival after axotomy. When 1 mg/kg body weight MK-801 is administered subcutaneously every day for 1-8 weeks to hemisected rats, cell death is prevented. Treatment with 0.5 mg/kg body weight MK-801 over the same time periods results in only partial rescue of axotomized neurons. Paradoxically, when 1 mg/kg MK-801 administration is restricted to the first week of an 8 week survival period, cell death in both the RN and CN is greatly exaggerated over the cell loss found in saline-treated animals. Withdrawal of 1 mg/kg MK-801 after 1 week of administration induces the loss of 92% of CN neurons, which is 63% greater than that occurring after axotomy alone. If, however, 1 mg/kg MK-801 is withdrawn after 2 weeks post-axotomy in the RN and 3 weeks postaxotomy in CN, all axotomized neurons survive. This rescue is found at 6 months postsurgery, the longest survival period studied, and therefore appears to be permanent. These results suggest that glutamatergic afferent input contributes significantly to the death of axotomized red nucleus and Clarke's nucleus neurons via NMDA receptors located on these neurons.

Dehydration and death during febrile episodes in the nursing home.

OBJECTIVE: To determine the incidence of early hypernatremic dehydration among residents of a nursing home care unit (NHCU) presenting with significant febrile episodes (FE). DESIGN: Prospective cohort analytic study. FE were defined as temperature (T) > 100 degrees F oral (o) or 101 degrees F rectal (r) for > or = 24 hours. SETTING: NHCU in a Veterans Administration hospital. PATIENTS: A total of 130 residents of the NHCU were monitored for FE during a 4-month study period. MAIN OUTCOME MEASURES: Blood urea nitrogen (BUN)/creatinine (Cr) (abnormal > or = 25) and serum sodium (Na)(abnormal > or = 146 mmol/L) were drawn within 24-48 hours of the onset of all FE; documentation of impaired oral intake (OI) by staff; necessity of transfer to acute medical wards and mortality were recorded. RESULTS: There were 48 FE among 42 residents (39 M, 3 F; mean age 75 +/- 11.3). Maximum recorded T during the FE ranged from 100.1 degrees F-102.2 degrees F o and 101.2 degrees F-105.3 degrees F r. Laboratory values were available for 40/48 FE. Twenty-three percent (9/40) had elevated BUN/Cr ratios, 25% (10/40) had elevated serum Na, and 12.5% (5/40) had both. In patients noted to have impaired OI (n = 11) as documented by staff, increased serum Na or BUN/Cr ratio was observed in 82% (9/11). A random control group of 37 nonacutely ill, nonfebrile NHCU residents (33 M, 4 F; mean age 75 +/- 10.1) having routine annual laboratory tests revealed only 1 resident (age 95) with an elevated Na of 146 and BUN/Cr ratio of 26 and 1 resident with an increased BUN/Cr ratio of 28. None of the controls had any staff documentation of impaired OI. Of the 5 deaths in the febrile group with laboratory data (total deaths = 6; 14%), 100% had either elevated serum Na and/or elevated BUN/Cr ratios, and 80% (4/5) had both. Comparing the febrile group with controls, BUN/Cr ratios were found to be significantly elevated in the febrile group (P < 0.05). Serum sodium values were also significantly elevated in the febrile group (P < 0.01). CONCLUSIONS: Staff documentation of impaired OI was highly associated with either elevated serum Na or increased BUN/Cr ratios. These data show that many older NHCU patients with significant fevers often have early impaired OI and laboratory evidence of dehydration. These data indicate that staff should institute appropriate monitoring for dehydration at the time of earliest detection of fever in this population.

Microglial invasion and activation in response to naturally occurring neuronal degeneration in the ganglion cell layer of the postnatal cat retina.

Retinae of kittens between postnatal (P) days 2 and 10 were examined for the presence of degenerating neuronal profiles, normal nucleoli and microglia. Comparison of the numbers of degenerating profiles with numbers of axons lost from the optic nerve suggest that the majority of these profiles result from the degeneration of retinal ganglion cells. Analysis of local densities of the different profiles revealed different rates of cell loss, occurring at different times in central and peripheral retina. The period of rapid cell loss occurred between P2 and P3 in central retina compared to between P8 and P10 in peripheral retina. At both locations, these periods of rapid cell loss were accompanied by a decrease in the ratio of microglia to dying cells even though the absolute densities of microglia increased. However, calculation of the clearance times of cellular debris indicate that the speed of removal of degeneration products is greater during rapid cell loss, which suggests that cellular degeneration serves to activate the phagocytic process.

Recovery of frontal cortex-mediated visual behaviors following neurotrophic rescue of axotomized neurons in medial frontal cortex.

Unilateral lesions extending across the boundary region of visual and parietal cortex in adult rats result in the death of 20-35% of neurons in layers II-III of the caudal third of medial frontal cortex ipsilaterally, a neuron population labeled with 3H-thymidine on the 19th day of gestation (E19). Additionally, there is a consistent 15% loss of these labeled neurons in an area between 50% and 60% of the distance along the caudal-rostral extent of medial frontal cortex, an area that may function analogously to the frontal eye field of primates. All of these neurons are rescued from axotomy-induced death by delivering into the posterior cortex lesion cavity for 2 weeks a macromolecular fraction of culture medium conditioned by embryonic primordia of the frontal-occipital pathway (CM). Moreover, the rescue is apparently permanent, with normal numbers of these neurons present in CM animals 6-7 weeks after the neurotrophic factor is no longer being supplied exogenously. Behaviorally, control operates receiving a similarly prepared fraction of unconditioned medium are significantly impaired in the number of trials needed to learn two visual discrimination tasks. This deficit is attributable in part to a bias in erroneous responses to the side contralateral to the lesion. The error bias reflects a failure to inhibit repeated incorrect responding contralaterally. In contrast, the CM animals learn both visual tasks in a normal number of trials and have no contralateral error bias. Rather, all CM animals have an contralateral error bias. Rather, all CM animals have an ipsilateral error bias (interpreted as an unmasking of the contralateral neglect expected after a parietal cortex lesion).(ABSTRACT TRUNCATED AT 250 WORDS)

Rescue of both rapidly and slowly degenerating neurons in the dorsal lateral geniculate nucleus of adult rats by a cortically derived neuron survival factor.

We investigated the death of dorsal lateral geniculate nucleus (dLGN) neurons after lesions to the visual cortex of adult rats and the effects of supplying target-derived neurotrophic molecules to the lesion cavity. The neurotrophic factor is retrieved from cocultures of the embryonic primordia of the geniculocortical pathway and its survival promoting properties for different populations of dLGN neurons (based on their time of origin) have been documented in previous studies of neonatal rats with occipital cortex lesions. In the present study, rats were exposed to [3H]thymidine on E14 or E15/16 to label either earlier or later generated dLGN neurons. When animals were at least 45 days old we made discrete lesions to the principal projection zones in area 17 of these two dLGN populations. Counts of surviving labeled cells show a relatively rapid death of E15/16 dLGN neurons in control animals, with a maximal loss by 2 weeks postlesion. The death of E14 dLGN neurons is more protracted, with a maximal loss by 2 months postlesion. A 2-week infusion of the CM fraction rescues the majority of the neurons that would otherwise die in both populations compared to the controls which receive a similarly prepared fraction of unconditioned medium. Moreover, this CM fraction can sustain E14-generated dLGN neurons up to 6 weeks after the neurotrophic factor(s) is no longer being supplied exogenously. Thus the rescue of axotomized adult dLGN neurons appears to be permanent, at least for the early generated population. These findings are consistent with the idea that target-derived molecules have a role in the survival of mature neurons, as they are known to have for developing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain.

Brain macrophages and microglia play important roles in central nervous system (CNS) development, especially during regressive events in which particular neuronal and glial constituents are eliminated. The purpose of this study is to provide a complete map of brain macrophage and microglia distribution in all regions of the neuraxis from birth to sexual maturity. We have utilized morphology and immunostaining with the specific antibodies OX-42 and ED1 to distinguish between brain macrophages and microglia. Brain macrophages are large, round cells, 10-15 microns in diameter, with few or no cytoplasmic processes; these cells are ED1- and OX-42-immunopositive. Microglia have small cell bodies with numerous, ramified cytoplasmic processes. These cells are OX-42-positive, and ED1-negative. We found a specific pattern of distribution of brain macrophages, targeting specific cortical and subcortical areas transiently, including developing fiber tracts. These cells disappeared completely by the third postnatal week. In contrast, OX-42-positive microglia exhibited a gradual increase in number and were distributed uniformly throughout gray matter and within white matter tracts. These cells remain in the adult CNS, constituting the resident microglia population. We suggest that these two distinct phagocytic cell populations perform unique functions in the developing brain, including remodeling of restricted CNS areas by brain macrophages that is part of a normal morphological process.

Brain macrophages and microglia respond differently to lesions of the developing and adult visual system.

Traumatic injury in the brain usually results in rapid degeneration of neuronal elements and a response by peripherally derived macrophages (brain macrophages, BMOs) and resident microglia. One intriguing result of lesions performed in the developing brain as compared to lesions of the mature brain is the faster resolution of the cellular debris and the absence of significant scarring. The purpose of this study was to examine the response of BMOs to induced cell death distant to the lesion site and to investigate possible differences in the responding phagocytic populations (BMOs versus microglia) following lesions in neonates and adults. Ablation of the visual cortex at birth results in very rapid retrograde degeneration and removal of neurons of the dorsal lateral geniculate nucleus (dLGN) within a few days. Lesions to the visual cortex of adult rats also induce neurons within the dLGN to die, but these cells do so over a much more protracted time course. Utilizing differences in morphology and immunocytochemical staining with the monoclonal antibodies ED1 and OX-42 to distinguish between BMOs and microglia, we found that in the developing CNS, BMOs are signalled rapidly and specifically to the location of induced cell death. Microglia are not involved in this response. As might be expected, the temporal response in the adult is much more protracted. In contrast to the developing brain, microglia and not macrophages are the predominant responding cell class after the adult lesion. The data suggest that these are distinct populations of phagocytic cells that respond to brain damage during development and in the adult, which may be critical in modulating the resolution and growth response after injury.

Anti-neutrophil cytoplasm antibodies in rheumatoid arthritis.

Anti-neutrophil cytoplasm antibodies (ANCA) occur occasionally in rheumatoid arthritis (RA), but their incidence and clinical significance have been unclear. In this study we have investigated 58 patients with RA. In 22 patients the disease was inactive and the remaining 36 with active disease were further subdivided into those without clinical evidence of vasculitis (26), those with cutaneous vasculitis (8) and those with systemic vasculitis (2). ANCA were demonstrated by indirect immunofluorescence in 10 of the 58 patients (17%). While both perinuclear (pANCA) and cytoplasmic (cANCA) staining were detected, pANCA were more common (70%). Neutrophil-specific anti-nuclear antibodies (ANNA) were demonstrated in a further eight sera (14%) and ANA were detected on Hep-2 cells in 30 of the 58 sera (52%). ELISAs for the detection of anti-myeloperoxidase and anti-elastase antibodies were then established. Five sera with pANCA and five that contained ANNA were negative for both anti-myeloperoxidase and anti-elastase antibodies, suggesting other as yet unidentified cytoplasmic antigens as the target molecules. However, anti-myeloperoxidase or anti-elastase antibodies were found in four sera that had homogeneous or speckled ANA on both Hep-2 cells and neutrophils. One serum contained both antibodies. The presence of ANCA detected by indirect immunofluorescence or of anti-myeloperoxidase or anti-elastase antibodies in these patients with RA was not associated with disease activity nor with the demonstration of cutaneous vasculitis or renal disease (P NS). A possible association with systemic vasculitis remains to be confirmed. There is an incomplete correlation between indirect immunofluorescence patterns and antibody specificity in ELISA systems.

Different populations of dorsal lateral geniculate nucleus neurons have concentration-specific requirements for a cortically derived neuron survival factor.

A macromolecular fraction of conditioned culture medium (CM) derived from explant cocultures of embryonic rat posterior cortex and caudal thalamus is able to support the survival of neurons in the dorsal lateral geniculate nucleus (dLGN) of newborn rats following ablation of dLGN cortical target areas. In the present study we tested whether the survival-promoting activity of this target-derived neurotrophic agent was concentration dependent and whether different subpopulations of dLGN neurons were equally responsive. With the starting concentration of the CM fraction designated X, increasing concentration results in a progressive falloff in trophic activity so that at 200X overall dLGN survival is similar to that seen in unconditioned medium (UM) controls. In contrast, diluting the fraction produces an increase in activity until maximal survival is achieved at 0.2X. Further dilutions result in a decline in trophic activity until control values are reached at 0.001X. Two populations of neurons within the dLGN, defined by their time of origin, respond in a specific manner to the different concentrations. Neurons generated during the early stages of neurogenesis (E14) have maximal survival (25.8%) at 0.05X, whereas those neurons generated later (E15/16) are maximally supported (30.7% survival) at 10X, a 200-fold difference in concentration. While it is possible that separate neurotrophic and neurotoxic molecules exist for each of these populations of dLGN neurons, the most parsimonious interpretation of the data is that a single cortically derived neurotrophic factor exists whose production is strictly controlled during development to achieve maximal effect on different populations of thalamic neurons that may be functionally distinct.

Rapidly progressive hip osteoarthrosis--an unusual presentation of ochronosis.

A case of rapidly evolving osteoarthrosis of the right hip in a 65 year-old woman, as the presenting feature of ochronosis is described and literature reviewed. This unusual presentation may have been precipitated by an acute mechanical overload.

Neurotrophic and behavioral effects of occipital cortex transplants in newborn rats.

Cell suspensions of embryonic occipital cortex were transplanted into newborn rats with large unilateral visual cortex lesions. When the animals were adults, they were tested on a difficult visual discrimination, and subsequently their brains were analyzed for possible neurotrophic effects of the transplants on nonvisual cortical areas which normally form connections with the occipital cortex. Behaviorally, animals with lesions and transplants learn to discriminate between columns and rows of squares at a rate which is identical to normal rats while animals with lesions and no transplants are impaired. Volume and cell-density measures show that the transplants also rescue neurons in cortical area 8 that would normally degenerate following the cortical lesion. No such neurotrophic effect of the transplants is found in cortical area 24 or area 17 contralateral to the lesion. In rats with lesions and no transplants, there is a significant correlation between the amount of area 8 remaining after the lesion and trials to criterion on the columns-rows discrimination, a relationship that does not exist in transplant animals because of their normal learning curve and the consistent sparing of area 8. Injections of HRP into the visual cortex contralateral to the lesion result in variable numbers of labeled cells within the transplant. However, there is no consistent relationship between the number of transplant cells which project to the opposite hemisphere and learning of the discrimination. It is suggested that the learning deficit following the lesion is largely attentional and that the sparing of cortical area 8 (which in rats may include the analog of the frontal eye fields present in the primate cortex) contributes to the sparing of function.

Diffusible proteins prolong survival of dorsal lateral geniculate neurons following occipital cortex lesions in newborn rats.

Removal of the occipital cortex in newborn rats results in the rapid and nearly complete degeneration of the dorsal lateral geniculate nucleus (dLGN) in 5 days. In previous studies we have shown that transplants of embryonic posterior cortex neurons, which are allowed to develop in culture for 5 days prior to transplantation into the site of the lesion, prolong the survival of a particular population of host dLGN neurons for an additional week. In this study we tested the possibility that the transplant cells synthesize diffusible proteins which are responsible for this neurotrophic effect. Culture medium conditioned by explants of embryonic occipital cortex and diencephalon was concentrated by vacuum dialysis or ultrafiltration through membranes with at least a 10-kDa cut-off. This concentrated medium was loaded into polyacrylamide or sodium alginate gels which were then implanted into the cavity of the lesion. Five days after implantation, the alginate-conditioned-medium implants result in a 3-fold increase in dLGN survival compared to unconditioned medium controls, while a two-fold increase in survival of the nucleus is found with the polyacrylamide-conditioned-medium implants. Proteolysis of the conditioned medium eliminates all neurotrophic activity. The results suggest that the death of dLGN neurons following the cortical lesion is due to the loss of diffusible proteinaceous neurotrophic factors--factors that may operate during normal in vivo development of the geniculocortical pathway.

Specific neurotrophic interactions between cortical and subcortical visual structures in developing rat: in vitro studies.

We investigated the influence of different subcortical structures on the survival of specific populations of occipital cortex neurons developing in vitro. Explants of embryonic day 14-15 (E14-15) rat cortex were cultured for 5 days with explants of either diencephalon or optic tectum or another occipital cortex explant. Stereological analysis of the explants revealed that after 5 days in vitro (5 DIV) all the cortical explants contained equal proportions of healthy neurons, glia, neuropil, and degenerating profiles, regardless of the culturing conditions. In order to determine if different neuronal populations survived preferentially in the cortical explants as a result of the presence of potential target or afferent structures, we used HRP filling and 3H-thymidine labeling techniques. Specific differences in the morphology of the cells and their time of origin are found in the cortical explants. In the cortical explants cocultured with diencephalon (Cx + D) the cortical cells that survive tend to be round with small cross-sectional areas and have few neurites. These cells are generated late in the culturing period. The surviving cortical neurons in the cortex plus tectum (Cx + T) cultures are larger--many with a pyramidal-shaped soma and several neurites. These cells are generated earlier in vitro. The cortex cultured with other cortex (Cx + Cx) gives values intermediate to the Cx + D and Cx + T cultures. The results of these experiments suggest that there are diffusible trophic factors that arise from subcortical structures that selectively support the survival of neuron populations in the developing neocortex.

Specific neurotrophic interactions between cortical and subcortical visual structures in developing rat: in vivo studies.

The specificity of trophic interactions in the rat visual system is investigated in vivo by using a combination of tissue culture and CNS transplantation methods. In a companion paper (Repka and Cunningham: '87) we showed that explants of embryonic day 14 (E14) occipital cortex are biased to contain different cortical cell populations depending on whether the explants develop in culture with diencephalon or with optic tectum. In this study we transplanted these precultured cortical explants into the cavity created by a lesion of the occipital cortex in newborn rats and then measured the neuron-occupied volume and the numbers of thymidine-labeled cells in the surviving ipsilateral dorsal lateral geniculate nucleus (dLGN) of the host rats. The results were compared to animals with lesions but no transplants, animals with transplants of E14 cortical tissue that had not been precultured, and animals with cerebellar transplants that had been similarly precultured either with other cerebellar tissue or with diencephalon. At 5 days postlesion, both the largest dLGN volume and the greatest number of labeled dLGN neurons survive in animals with cortical transplants precultured with diencephalon or other cortex. The surviving dLGN neurons that are rescued by these transplants are generated on E15 or E16, a period that corresponds to the latter part of geniculate neurogenesis. Relatively few cells generated on E14 survive in any group of animals. Furthermore, animals with all types of cortical transplants have significantly larger volumes of surviving dLGN than animals with either lesions only or cerebellar transplants.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase II evaluation of dibromodulcitol and actinomycin D, hydroxyurea, and cyclophosphamide in previously untreated patients with malignant melanoma.

In this Eastern Cooperative Oncology Group (ECOG) phase II study, dibromodulcitol (DBD) and a combination of actinomycin D, hydroxyurea, and cyclophosphamide (AHC) were compared with methyl-CCNU, the current ECOG standard, in patients who had received no prior chemotherapy for disseminated malignant melanoma. The response rates were 6% (3/50) for AHC, 9% (3/34) for DBD, and 14% (7/49) for methyl-CCNU. Median survival times were 4, 5, and 6 months, respectively. Neither regimen appears to offer any advantage over methyl-CCNU as front-line therapy for patients with disseminated melanoma.