- Email: [email protected]
New approaches to the study of central nervous system function. Immune-nervous system interactions and cell culture
Neurobiology of Aging, Vol. 9, pp. 763-765. © Pergamon Press plc, 1988. Printed in the U.S.A.
0197-4580/88 $3.00 + .00
New Approaches to the Study of Central Nervous System Function. Immune-Nervous System Interactions and Cell Culture D A V I D G. M O R G A N A N D M A R C I A N. G O R D O N
Andrus Gerontology Center, University of Southern California, Los Angeles, CA 90089-0191 and Mental Retardation Research Center, University o f California, Los Angeles, CA 90024
The paper by Lal and Forster is discussed with reference to future experiments which might provide insight into mechanisms regarding their exciting data that circulating brain reactive antibodies may cause learning deficits. The paper by Azmitia et al. on cell culture techniques is discussed with respect to the types of studies in which culture systems have proven most valuable in the past, and should continue to do so in the future.
OVER the last 25 years, the field of neuroscience has evolved into an independent, biological research discipline. Neuroscience crosses most biological discipline boundaries, and traditionally includes anatomists, behaviorists, chemists, and physiologists. Recently, molecular geneticists have been added to the fold, and this section includes two other disciplines, immunology and cell culture. Every possible approach to solving the riddles of the nervous system is welcome. We provide here some directions in which we feel these disciplines may aid in unders.tanding neural function, and some caveats concerning the use of these approaches. The paper by Lal and Forster describes an intriguing series of results focusing on immune system modulation of central nervous system function. The authors find a striking correlation between age-related increases in brain reactive antibodies (BRAs) and learning deficits in a series of mouse strains (based on group mean data). These initial data could be interpreted as two independent consequences of agerelated processes occurring generally .in these organisms (i.e., not causally related). However, the immune system transfer experiments indicate some causal relation between the BRAs and the retarded acquisition of one-way avoidance tasks. This leaves two important questions which remain to be answered; what antigens are these BRAs directed towards, and how do they gain access to these antigens within the brain parenchyma? Experiments to answer the first question appear straightforward. The authors appropriately indicate that examination of the BRA titer and acquisition performance of individual animals need to be performed. This will serve to tighten the correlation based on pooled age-group and strain data. However, simultaneous examination of Western blots using the sera from the learning deficient animals may reveal a subset of antibodies which are most critical in inducing these learning deficits. A first approach might examine strips of brain proteins electrophoresed on SDS-polyacrylamide gels, with each strip incubated with serum from a different animal. The discovery of high titers against specific molecular weight regions common to all learning deficient animals may suggest specific proteins which are involved in these phenomena, and implicate mechanisms by which BRAs might interfere with neuronal function. The second question, how these antibodies gain access to
the brain to disrupt learning, is not as readily amenable to experimentation. The authors point out that leaks in the blood-brain barrier may accumulate with age, but data concerning this possibility are equivocal. A second possibility is that neurons which have processes extending beyond the blood-brain barrier may actively take up and transport antibodies via retrograde axonal transport. Evidence supporting this possibility was obtained by Fabian and Ritchie (1986). These authors stained rat brain sections for the presence of rat IgG and found a distinct staining pattern (although the age of the rats is not presentebd in this manuscript, we assume they were young adults). IgG was found in two general areas; those which are known to possess a poor blood-brain barrier such as the area postrema and hypothalamus, and those areas which contain motor neurons projecting to the periphery (ventral horn neurons; neurons in the cranial nerve motor nuclei). Retrograde axonal transport from terminal zones to somatic regions is a general phenomenon of all neurons. This transport can be both specific, as for nerve growth factor, and nonspecific. This nonspecific retrograde transport is the basis of many hodological (fibertract tracing) studies with peroxidase and other labels. Viruses (such as Herpes) may also use this mechanism for access to neuronal cell bodies where they can integrate with the genome. Once inside the CNS, the antibodies may possibly distribute to other cell types via transneuronal transport. If the general concept that in the CNS a given neuron is only three or four synapses away from any other neuron is correct, then these antibodies may gain access to specific cell targets and concentrate there. A second possibility is that the filtering of blood to form cerebrospinal fluid (CSF) by the choroid plexus is slightly defective. This might allow circulating IgG access to the brain parenchyma through the ependymal cells lining the ventricles (ependymal cells were also IgG positive in the study of Fabian and Ritchie). A third question is how brain antigen presentation to the immune system occurs? Answers to this question may be even more elusive than the first two, but may ultimately be more important in preventing the loss of learning capacity produced by the BRAs. One possibility is that the antigens are actually present in peripheral portions of central neurons, or are common to both central and peripheral
7fi'~
764 neurons. Peripheral nerves have a weak type of blood-brain barrier called the blood-nerve barrier. If the immune system can penetrate this barrier, it would be presented with a series of axonal proteins which may not be recognized as self. These may then induce an immune reaction leading to the formation of antibodies directed against brain antigens. Perhaps in the Western blot studies suggested above, sera should also be tested for reactivity towards peripheral nerve proteins as well. As researchers studying the baffling problem of Alzheimer-type dementia, we are aware of several studies reporting the presence of BRAs in Alzheimer's disease patients. One question concerning these studies is whether the presence of these BRAs is etiologic in the disease process, or a symptom of general neuronal damage, and local leakage of brain proteins into the circulation. It would be of interest to determine if neuronal damage (such as that produced by peripheral kainic acid or other systemically administered neurotoxins) causes an increase in the formation of BRAs, and subsequently learning deficits. It is known that brain seizures do cause transient opening of the blood-brain barrier [8]. Such openings may conceivably be general occurrences in regions of high neuronal activity, allowing IgG in and brain antigens out of the CNS, thus answering both the second and third questions. This finding of immune system dysfunction leading to learning deficits is made particularly salient by the presence of dementia symptoms in many AIDS patients. Some mystery surrounds the mechanism by which the AIDS virus might produce dementia symptoms since it does not appear to infect neurons. The primary cells containing AIDS virus in the CNS are macrophages and microglia [7], and possibly astrocytes [5]. Astrocytes may play a critical role in this process because they are thought to induce endothelial cells around brain capillaries to produce the tight junctions responsible for the blood-brain barrier. Dysfunction in these astrocytes may break down the blood-brain barrier, allowing brain antigen presentation to the immune system, formation of BRAs, and subsequent neuronal dysfunction. While speculative at present, the results of Lal and Forster suggest that BRAs should be examined in AIDS patients as a potential source of the dementia symptoms found in these patients. One final comment concerns the selectivity of these BRAs for the acquisition phase (learning) of the one-way avoidance task, but not the retention phase (memory) based on the strain comparisons. This finding should be confirmed in other learning tasks and in the immune system transfer studies. However, if this initial finding is correct, these B R A s may be important tools to study the different steps involved in the encoding of memory as distinct from the acquisition of the task per se. The review by Azmitia et al. on the relative merits of cell culture in studies of aging and neurodegeneration requires less commentary because most concerns were presented in the review. We share with these authors a healthy skepticism of the experiments describing the effects of various ions and molecules on neuron survival in vitro. Azmitia et al. point out that neuron-promoting effects of most factors are dosedependent (often with inverted U functions), environment (culture condition) specific, and apparent only at developmentally relevant time frames. We are aware of a number of other papers which these authors did not have time to discuss, in which different concentrations of factors, modifications of culture conditions, or use of cells of different developmental ages resulted in contradictory findings. For
M O R G A N A N D GORDON example, while hydrogen peroxide can be neurotoxic (as discussed by Azmitia et al.), it is also neurotrophic when supplied at lower concentrations [2]. The presence of other cell types in the culture is also an important consideration. Brenneman et al. [4] found that vasoactive intestinal polypeptide had neurotrophic effects in neuron/astrocyte co-cultures, but not in astrocyte-free cultures, implying an indirect astrocyte-mediated effect. Hormonal synergy is common in cell culture paradigms; individual hormones may exert little growth-promoting effects in isolation, but substantial effects when added in combination [1,3]. Serum addition actually defeats one of the primary advantages of in vitro systems, which is rigorous control of the cell milieu, and may influence responsivity to neurotrophic agents [6]. Another interpretive problem with neuron cell culture is that most cultures are derived from embryonic, or immediately postnatal animals. Indeed, it appears that the neurons which survive most readily in culture are those which have just stopped dividing and are starting to differentiate, but have not yet extended axonal and dendritic processes which may be severed during the culture procedure. As a result, most cultures consist of neurons which are characterized as partially differentiated. We feel it is important to distinguish between factors which support differentiation from factors which support neuron survival, and these issues may become confused in culture systems unless careful choice of dependent measures is made. It is important to consider that normal development in the nervous system is accompanied by loss of about half of the neurons. Thus, neurodegeneration accompanying aging and the loss of neurons in culture may represent fundamentally different processes. It is our opinion that the strength of cell culture in the past has been to study the mechanism of cell functions which were originally described in vivo. Under these circumstances, the ability to strictly control oxygen and glucose levels, to effectively label macromolecule precursor pools, and to tightly regulate the concentrations of various modifying substances allow the mechanism of cell functions to be dissected. However, descriptive studies of the culture system itself are probably of limited value in predicting in vivo phenomena. One example of this problem is obliquely referenced by Azmitia et al. Nerve growth factor has been demonstrated by a number of authors to be produced by astrocytes in culture (referenced in Azmitia et al.). However, direct examination of the cells possessing N G F RNA in vivo by in situ hybridization indicates an exclusively neuronal origin, at least in hippocampus. It has been rumored that any cell type left in culture for a long enough time will express N G F (Franz Hefti, personal communication). Obviously, a noncritical interpretation of the astrocyte culture literature may lead to misimpressions of the in vivo condition. We do not mean to imply that descriptive studies in culture systems are not without merit. They may point to important cell functions which can be tested in vivo. We merely suggest, as pointed out by Azmitia et al., that such results be confirmed in vivo before they are accepted as generally applicable. We agree with Azmitia et al. that a second strength of the culture system is the use of cultures as an end point dependent variable in isolating potential in vivo derived neurotrophic factors (or toxins). Other assays for assessing the active fi'actions either do not exist or are extremely tedious and expensive, or not quantifiable. Primary characterization of neurotrophic potential in culture systems is an important adjunct to tests in vivo, yet, ultimately, in vivo tests must be performed to confirm the neurotrophic influence.
COMMENTARY
765
REFERENCES 1. Aizenman, Y. and J. deVellis. Brain neurons develop in a serum and glial-free environment: Effects of transferrin, insulin, insulin-like growth factor I, and thyroid hormone on neuronal survival, growth and differentiation. Brain Res 406: 32-42, 1987. 2. Boioga, L., J. Sharma, D. Dahi and E. Roberts. Buffers and H202 reduce neuronal death and/or enhance differentiation of neurons and astrocytes in dissociated mouse brain cultures. Brain Res 411: 282-290, 1987. 3. Bottenstein, J. E. and G. H. Sato. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci USA 76: 514-517, 1979. 4. Brenneman, D. E., E. A. Neale, G. A. Foster, S. W. D'autremont and G. L. Westbrook. Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide. J Cell Biol 104: 1603-1610, 1987.
5. Epstein, L. G., L. R. Sharer, V. V. Joshi, M. M. Fojas, M. R. Koenigsberger and J. M. Oleske. Progressive encephalopathy in children with acquired immune deficiency syndrome. Ann Neurol 17: 488-496, 1985. 6. Furukawa, S., Y. Furukawa, E. Satoyoshi and K. Hayashi. Synthesis/secretion of nerve growth factor is associated with cell growth in cultured mouse astroglial cells. Biochem Biophys Res Commun 142: 395-402, 1987. 7. Koenig, S., H. E. Gendelman, J. M. Orenstein, M. C. DalCanto, G. H. Pezeshkpour, M. Yungbluth, F. Janotta, A. Aksamit, M. A. Martin and A. Fauci. Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 233: 1089-1093, 1986. 8. Ruth, R. E. Increased cerebrovascular permeability to protein during systemic kainic acid seizures. Epilepsia 25: 259-268, 1984.
Letters to the Editor W e e n c o u r a g e o u r readers to r e s p o n d to this review and a s s o c i a t e d p e e r c o m m e n t a r i e s in the form o f formal " L e t t e r s to the E d i t o r . " Relatively s h o r t letters with a specific p o i n t o r two are m o s t a p p r o p r i a t e . In all cases, the a u t h o r o f the original review o r c o m m e n t a r y will be given an o p p o r t u n i t y to r e s p o n d to the letter.
0197-4580/88 $3.00 + .00
New Approaches to the Study of Central Nervous System Function. Immune-Nervous System Interactions and Cell Culture D A V I D G. M O R G A N A N D M A R C I A N. G O R D O N
Andrus Gerontology Center, University of Southern California, Los Angeles, CA 90089-0191 and Mental Retardation Research Center, University o f California, Los Angeles, CA 90024
The paper by Lal and Forster is discussed with reference to future experiments which might provide insight into mechanisms regarding their exciting data that circulating brain reactive antibodies may cause learning deficits. The paper by Azmitia et al. on cell culture techniques is discussed with respect to the types of studies in which culture systems have proven most valuable in the past, and should continue to do so in the future.
OVER the last 25 years, the field of neuroscience has evolved into an independent, biological research discipline. Neuroscience crosses most biological discipline boundaries, and traditionally includes anatomists, behaviorists, chemists, and physiologists. Recently, molecular geneticists have been added to the fold, and this section includes two other disciplines, immunology and cell culture. Every possible approach to solving the riddles of the nervous system is welcome. We provide here some directions in which we feel these disciplines may aid in unders.tanding neural function, and some caveats concerning the use of these approaches. The paper by Lal and Forster describes an intriguing series of results focusing on immune system modulation of central nervous system function. The authors find a striking correlation between age-related increases in brain reactive antibodies (BRAs) and learning deficits in a series of mouse strains (based on group mean data). These initial data could be interpreted as two independent consequences of agerelated processes occurring generally .in these organisms (i.e., not causally related). However, the immune system transfer experiments indicate some causal relation between the BRAs and the retarded acquisition of one-way avoidance tasks. This leaves two important questions which remain to be answered; what antigens are these BRAs directed towards, and how do they gain access to these antigens within the brain parenchyma? Experiments to answer the first question appear straightforward. The authors appropriately indicate that examination of the BRA titer and acquisition performance of individual animals need to be performed. This will serve to tighten the correlation based on pooled age-group and strain data. However, simultaneous examination of Western blots using the sera from the learning deficient animals may reveal a subset of antibodies which are most critical in inducing these learning deficits. A first approach might examine strips of brain proteins electrophoresed on SDS-polyacrylamide gels, with each strip incubated with serum from a different animal. The discovery of high titers against specific molecular weight regions common to all learning deficient animals may suggest specific proteins which are involved in these phenomena, and implicate mechanisms by which BRAs might interfere with neuronal function. The second question, how these antibodies gain access to
the brain to disrupt learning, is not as readily amenable to experimentation. The authors point out that leaks in the blood-brain barrier may accumulate with age, but data concerning this possibility are equivocal. A second possibility is that neurons which have processes extending beyond the blood-brain barrier may actively take up and transport antibodies via retrograde axonal transport. Evidence supporting this possibility was obtained by Fabian and Ritchie (1986). These authors stained rat brain sections for the presence of rat IgG and found a distinct staining pattern (although the age of the rats is not presentebd in this manuscript, we assume they were young adults). IgG was found in two general areas; those which are known to possess a poor blood-brain barrier such as the area postrema and hypothalamus, and those areas which contain motor neurons projecting to the periphery (ventral horn neurons; neurons in the cranial nerve motor nuclei). Retrograde axonal transport from terminal zones to somatic regions is a general phenomenon of all neurons. This transport can be both specific, as for nerve growth factor, and nonspecific. This nonspecific retrograde transport is the basis of many hodological (fibertract tracing) studies with peroxidase and other labels. Viruses (such as Herpes) may also use this mechanism for access to neuronal cell bodies where they can integrate with the genome. Once inside the CNS, the antibodies may possibly distribute to other cell types via transneuronal transport. If the general concept that in the CNS a given neuron is only three or four synapses away from any other neuron is correct, then these antibodies may gain access to specific cell targets and concentrate there. A second possibility is that the filtering of blood to form cerebrospinal fluid (CSF) by the choroid plexus is slightly defective. This might allow circulating IgG access to the brain parenchyma through the ependymal cells lining the ventricles (ependymal cells were also IgG positive in the study of Fabian and Ritchie). A third question is how brain antigen presentation to the immune system occurs? Answers to this question may be even more elusive than the first two, but may ultimately be more important in preventing the loss of learning capacity produced by the BRAs. One possibility is that the antigens are actually present in peripheral portions of central neurons, or are common to both central and peripheral
7fi'~
764 neurons. Peripheral nerves have a weak type of blood-brain barrier called the blood-nerve barrier. If the immune system can penetrate this barrier, it would be presented with a series of axonal proteins which may not be recognized as self. These may then induce an immune reaction leading to the formation of antibodies directed against brain antigens. Perhaps in the Western blot studies suggested above, sera should also be tested for reactivity towards peripheral nerve proteins as well. As researchers studying the baffling problem of Alzheimer-type dementia, we are aware of several studies reporting the presence of BRAs in Alzheimer's disease patients. One question concerning these studies is whether the presence of these BRAs is etiologic in the disease process, or a symptom of general neuronal damage, and local leakage of brain proteins into the circulation. It would be of interest to determine if neuronal damage (such as that produced by peripheral kainic acid or other systemically administered neurotoxins) causes an increase in the formation of BRAs, and subsequently learning deficits. It is known that brain seizures do cause transient opening of the blood-brain barrier [8]. Such openings may conceivably be general occurrences in regions of high neuronal activity, allowing IgG in and brain antigens out of the CNS, thus answering both the second and third questions. This finding of immune system dysfunction leading to learning deficits is made particularly salient by the presence of dementia symptoms in many AIDS patients. Some mystery surrounds the mechanism by which the AIDS virus might produce dementia symptoms since it does not appear to infect neurons. The primary cells containing AIDS virus in the CNS are macrophages and microglia [7], and possibly astrocytes [5]. Astrocytes may play a critical role in this process because they are thought to induce endothelial cells around brain capillaries to produce the tight junctions responsible for the blood-brain barrier. Dysfunction in these astrocytes may break down the blood-brain barrier, allowing brain antigen presentation to the immune system, formation of BRAs, and subsequent neuronal dysfunction. While speculative at present, the results of Lal and Forster suggest that BRAs should be examined in AIDS patients as a potential source of the dementia symptoms found in these patients. One final comment concerns the selectivity of these BRAs for the acquisition phase (learning) of the one-way avoidance task, but not the retention phase (memory) based on the strain comparisons. This finding should be confirmed in other learning tasks and in the immune system transfer studies. However, if this initial finding is correct, these B R A s may be important tools to study the different steps involved in the encoding of memory as distinct from the acquisition of the task per se. The review by Azmitia et al. on the relative merits of cell culture in studies of aging and neurodegeneration requires less commentary because most concerns were presented in the review. We share with these authors a healthy skepticism of the experiments describing the effects of various ions and molecules on neuron survival in vitro. Azmitia et al. point out that neuron-promoting effects of most factors are dosedependent (often with inverted U functions), environment (culture condition) specific, and apparent only at developmentally relevant time frames. We are aware of a number of other papers which these authors did not have time to discuss, in which different concentrations of factors, modifications of culture conditions, or use of cells of different developmental ages resulted in contradictory findings. For
M O R G A N A N D GORDON example, while hydrogen peroxide can be neurotoxic (as discussed by Azmitia et al.), it is also neurotrophic when supplied at lower concentrations [2]. The presence of other cell types in the culture is also an important consideration. Brenneman et al. [4] found that vasoactive intestinal polypeptide had neurotrophic effects in neuron/astrocyte co-cultures, but not in astrocyte-free cultures, implying an indirect astrocyte-mediated effect. Hormonal synergy is common in cell culture paradigms; individual hormones may exert little growth-promoting effects in isolation, but substantial effects when added in combination [1,3]. Serum addition actually defeats one of the primary advantages of in vitro systems, which is rigorous control of the cell milieu, and may influence responsivity to neurotrophic agents [6]. Another interpretive problem with neuron cell culture is that most cultures are derived from embryonic, or immediately postnatal animals. Indeed, it appears that the neurons which survive most readily in culture are those which have just stopped dividing and are starting to differentiate, but have not yet extended axonal and dendritic processes which may be severed during the culture procedure. As a result, most cultures consist of neurons which are characterized as partially differentiated. We feel it is important to distinguish between factors which support differentiation from factors which support neuron survival, and these issues may become confused in culture systems unless careful choice of dependent measures is made. It is important to consider that normal development in the nervous system is accompanied by loss of about half of the neurons. Thus, neurodegeneration accompanying aging and the loss of neurons in culture may represent fundamentally different processes. It is our opinion that the strength of cell culture in the past has been to study the mechanism of cell functions which were originally described in vivo. Under these circumstances, the ability to strictly control oxygen and glucose levels, to effectively label macromolecule precursor pools, and to tightly regulate the concentrations of various modifying substances allow the mechanism of cell functions to be dissected. However, descriptive studies of the culture system itself are probably of limited value in predicting in vivo phenomena. One example of this problem is obliquely referenced by Azmitia et al. Nerve growth factor has been demonstrated by a number of authors to be produced by astrocytes in culture (referenced in Azmitia et al.). However, direct examination of the cells possessing N G F RNA in vivo by in situ hybridization indicates an exclusively neuronal origin, at least in hippocampus. It has been rumored that any cell type left in culture for a long enough time will express N G F (Franz Hefti, personal communication). Obviously, a noncritical interpretation of the astrocyte culture literature may lead to misimpressions of the in vivo condition. We do not mean to imply that descriptive studies in culture systems are not without merit. They may point to important cell functions which can be tested in vivo. We merely suggest, as pointed out by Azmitia et al., that such results be confirmed in vivo before they are accepted as generally applicable. We agree with Azmitia et al. that a second strength of the culture system is the use of cultures as an end point dependent variable in isolating potential in vivo derived neurotrophic factors (or toxins). Other assays for assessing the active fi'actions either do not exist or are extremely tedious and expensive, or not quantifiable. Primary characterization of neurotrophic potential in culture systems is an important adjunct to tests in vivo, yet, ultimately, in vivo tests must be performed to confirm the neurotrophic influence.
COMMENTARY
765
REFERENCES 1. Aizenman, Y. and J. deVellis. Brain neurons develop in a serum and glial-free environment: Effects of transferrin, insulin, insulin-like growth factor I, and thyroid hormone on neuronal survival, growth and differentiation. Brain Res 406: 32-42, 1987. 2. Boioga, L., J. Sharma, D. Dahi and E. Roberts. Buffers and H202 reduce neuronal death and/or enhance differentiation of neurons and astrocytes in dissociated mouse brain cultures. Brain Res 411: 282-290, 1987. 3. Bottenstein, J. E. and G. H. Sato. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci USA 76: 514-517, 1979. 4. Brenneman, D. E., E. A. Neale, G. A. Foster, S. W. D'autremont and G. L. Westbrook. Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide. J Cell Biol 104: 1603-1610, 1987.
5. Epstein, L. G., L. R. Sharer, V. V. Joshi, M. M. Fojas, M. R. Koenigsberger and J. M. Oleske. Progressive encephalopathy in children with acquired immune deficiency syndrome. Ann Neurol 17: 488-496, 1985. 6. Furukawa, S., Y. Furukawa, E. Satoyoshi and K. Hayashi. Synthesis/secretion of nerve growth factor is associated with cell growth in cultured mouse astroglial cells. Biochem Biophys Res Commun 142: 395-402, 1987. 7. Koenig, S., H. E. Gendelman, J. M. Orenstein, M. C. DalCanto, G. H. Pezeshkpour, M. Yungbluth, F. Janotta, A. Aksamit, M. A. Martin and A. Fauci. Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 233: 1089-1093, 1986. 8. Ruth, R. E. Increased cerebrovascular permeability to protein during systemic kainic acid seizures. Epilepsia 25: 259-268, 1984.
Letters to the Editor W e e n c o u r a g e o u r readers to r e s p o n d to this review and a s s o c i a t e d p e e r c o m m e n t a r i e s in the form o f formal " L e t t e r s to the E d i t o r . " Relatively s h o r t letters with a specific p o i n t o r two are m o s t a p p r o p r i a t e . In all cases, the a u t h o r o f the original review o r c o m m e n t a r y will be given an o p p o r t u n i t y to r e s p o n d to the letter.