Home care at a crossroad.

This decade's fastest growing segment of the health care industry, home health care, is facing a significant challenge as a result of changes in Medicare reimbursement included in the Balanced Budget Act of 1997. The implications could prove to substantially reshape the industry as well as many of its providers.

Immunohistological localization of IgG1, IgA and secretory component in the bovine mammary gland during involution.

Immunoperoxidase methods were used to localize secretory component, immunoglobulin A and immunoglobulin G1 in mammary tissue from dairy cows. In lactating tissue, immunostaining for immunoglobulin A and secretory component was observed primarily in the luminal contents of alveoli. By day 2 of involution, alveolar epithelial cells stained for both immunoglobulin A and secretory component. Staining of alveolar epithelial cells for immunoglobulin A and secretory component continued throughout the period of mammary involution. No staining for secretory component was observed in the interalveolar stromal area. Immunoglobulin G1 immunostaining was localized primarily in the interalveolar areas in lactating tissue, but was localized at the apical and basolateral surface of alveolar cells on day 2 of involution. In contrast to immunoglobulin A, immunoglobulin G1 staining of epithelial cells did not persist and was primarily in the interalveolar areas by day 4. These results suggest that an increased localization of immunoglobulin G1 in bovine mammary epithelial cells may occur transiently in early involution, while an increase in immunoglobulin A and secretory component localization in epithelial cells persists throughout involution.

Age and previous lactations as factors in the amount of bovine colostral immunoglobulins.

Blood and colostrum samples were obtained from 87 dairy cows in five lactation groups and analyzed for immunoglobulins G1, G2, M, and A. The five groups ranged from cows in first lactation, about 30 mo of age, to cows in fifth or more lactation, about 84 mo of age. Compared to older groups, blood serum of cows in first lactation contained less G1. Cows in first lactation also produced less total colostrum containing less total G1, G2, and M. Immunoglobulin G1 comprised over two-thirds of the immunoglobulins in the colostrum of all groups. Older cows had more immunoglobulin G1 in their colostrum with a tendency toward a higher ratio of G1 to G2. Amount of immunoglobulin A was constant through all lactations. After a rise in the second lactation, total amount of immunoglobulins G2 and M tended to level off. Total immunoglobulin G1 tended to reach a maximum in the third or fourth lactation, almost doubling in amount compared to the first lactation. Age and number of lactations are factors correlated with amounts of these immunoglobulins in colostrum.

Immunohistochemical localization of IgG1 and IgG2 in prepartum and lactating bovine mammary tissue.

The differential distributions of IgG1 and IgG2 were determined in prepartum and lactating bovine mammary tissue by indirect immunofluorescence. IgG1 was found predominately within the alveolar epithelial cells and lumens of prepartum tissue whereas IgG2 was largely confined to the stromal area surrounding the alveoli. Both IgG subclasses were confined predominately to the stroma in lactating tissue. Few IgG containing stromal cells were readily distinguished in any of the mammary tissue used in this study.

Subcellular localization of pyrimidine synthesis enzymes in bovine mammary tissue.

Subcellular localization of enzymes of the pyrimidine biosynthetic pathway was determined in lactating bovine mammary tissue. Aspartate transcarbamylase was associated with a membrane portion of the microsomal cell fraction. Dihydroorotate dehydrogenase w3as in the mitochondrial fraction with no evidence for a cytosolic enzyme requiring pyridine nucleotides. Orotate phosphoribosyltransferase was in the cytosol of the cell. The significance of these findings to other mammalian cells and orotic acid accumulation in bovine milk is discussed.

Immunoglobulin production and transport by the mammary gland.

In origin immunoglobulins in mammary secretions are both humoral, arising from the blood stream, and local, arising from production by plasmacytes in the mammary gland. The relative importance of each of these sources varies between species. In some species (human, rabbit, etc.), the transfer of maternal immunoglobulins to the blood stream of the neonate occurs in utero across the placenta or yolk sac membrane. In other species, including ruminants, transfer of maternal immunoglobulins to the neonate occurs exclusively via the colostrum. Both in utero and colostral routes of transfer are operative in other species. The concentration and class of immunoglobulins in the colostrum and milk of a species reflect the route and origin of the immunoglobulins. Immunoglobulins transferred in quantity in utero or via the colostrum are mainly of the IgG class. Immunoglobulins locally produced by plasmacytes located adjacent to the secretory epithelium and in the mammary secretions are largely of the IgA and IgM classes. The bovine transfers large amounts of IgG immunoglobulins, and IgG1 in particular, from the blood stream across the mammary barrier into colostrum (and milk) by a specific transport mechanism. Bovine colostrum and milk also contain much smaller amounts of locally produced IgA and IgM.

Endogenous production of immunoglobulin IgG1 in newborn calves.

There is a decrease in the specific activity of labeled IgG1 of serum over 3 wk following the feeding of iodine-125 labeled immunoglobulin IgG1 in colostrum to calves at birth. This decrease indicated the appearance of new IgG1 from some source. To determine if this new IgG1 came from endogenous production in the calf or from continued small amount of intestinal absorption from milk, labeled IgG1 was added to normal milk and fed to calves of various ages up to 3 wk after an initial feeding of colostrum at birth. Labeled IgG1 was also added to colostrum fed to calves at birth, and the calves were maintained on a normal milk diet or fed a synthetic milk diet. Determination of iodine-125 in the serum protein fractions of these calves indicated that there was no apparent intestinal absorption of labeled IgG1 from the milk in the period from 2 days to 3 wk. Furthermore, comparable decreases occurred in the specific activity of labeled IgG1 in serum in the calves fed the labeled IgG1 in colostrum at birth and subsequently maintained either on a diet including milk or on the synthetic milk diet devoid of IgG1. The results support the conclusion that the origin of new IgG1 in the calf after about 36 h and up to about 3 wk of age arises from endogenous production at a rate of about 1 g of IgG1 per day.

Biosynthesis and secretion of milk proteins: a review.

Recent years have seen a great increase in the knowledge and understanding of milk proteins. Arising from several origins including the blood stream and various cellular sources, many of the proteins found in milk are products of the secretory cells directly involved in the synthesis and secretion processes of various milk components. The lactation-specific proteins present in major amounts are synthesized in the rough endoplasmic reticulum (RER) under genetic control and undergo further post-translational modifications in their secretory route from the RER through the Golgi apparatus and secretory vesicles before ejection into the lumen with other milk components. Various molecular aspects of these mechanisms and their control are now understood, but many remain to be described.

The dairy goat as a model in lactation studies.

The dairy goat has been used widely in lactation research due in large part to availability, convenience in size and handling, economic considerations, and similarity to the cow as a ruminant species in general and metabolic processes. Considerable basic knowledge of lactation relating to all species has been derived from studies with goats. Although the gross composition of goat and cow milk is similar, significant differences reflect differing synthetic functions. Caution is necessary in using the goat as a model for the dairy cow where differences occur. Some are obvious such as the gross structure of the mammary gland and differing milk constituents. Others are more subtle, such as the susceptibility to metabolic diseases associated with lactation and differing rates of metabolism affecting transfer of dietary and administered materials into milk.

Isolation and partial characterization of rate casein proteins.

Casein was isolated from rat milk by high speed centrifugation. Polyacrylamide disc gel electrophoresis of the whole casein yielded three major protein zones designated C.1, C.2, and C.3 in order of their decreasing electrophoretic mobility in the alkaline system. Zone 3 subsequently contained two possibly related bands, C.3.1 and C.3.2. The presence of phosphate in all four zones was indicated by staining and conformed by phosphorus-32 labeling studies. A glycoprotein character was indicated by all zones. Separation of the constituents of rat casein by diethylaminoethyl-cellulose ion exchange chromatography yielded the same four major protein entities. Three milk-specific phosphoproteins unique to rat whey cluted from such columns in the same general region as the casein constituents but appear to be otherwise unrelated to the four major components of micellar casein. Gel electrophoresis in sodium dodecyl sulfate systems yielded apparent molecular weight estimates of approximately 24,000 for C.1, 38,000 for C.2, and 28,000 for c.3.1 and c.3.2.

Purification and characterization of rat alpha-lactalbumins: apparent genetic variants.

Rat alpha-lactalbumin, from the milk of Fischer 344 (CDF) rats, was isolated and purified by a combination of gel filtration and diethylaminoethyl-cellulose ion exchange chromatography. Three electrophoretically distinct proteins had alpha-lactalbumin activity. Staining for carbohydrate indicated that at least two of the three forms were glycoproteins. The low molecular weight protein fraction from the wheys of two additional strains of laboratory rat were compared to ascertain whether the composition of this fraction was common in the divergent strains. Outbred Wistar and Long-Evans dams yielded wheys containing up to six forms of alpha-lactalbumin. Either one or both of two groups of three alpha-lactalbumins were in a given milk sample. The two groups of three alpha-lactalbumins appear to represent two genetic variants upon which is imposed a polymorphic character. All forms of alpha-lactalbumin, within and between strains, were immunologically identical.

Purification and partial characterization of a unique group of phosphoproteins from rat milk whey.

A group of four electrophoretically distinct but related proteins has been isolated from rat milk whey. These have been classified as P.1 to P.4 in order of their decreasing electrophoretic mobility in an alkaline buffer system. All four members of this group of proteins are immunologically identical apparently constitute a single protein which is differentially phosphorylated in the ratio of 3:2:1:0. Sodium dodecyl sulfate gel electrophoresis yielded constant estimates of molecular weight of approximately 20,700. Enzymatic dephosphorylation of purified P.1 generated the other three members of the group, demonstrating the commonality of the peptide component. This group of proteins in total makes a significant contribution of the total whey proteins being approximately 5 mg/ml in whole rat milk in the approximate proportion of 1:1.5:2:1.5. The P.1, P.2, and P.3 were isolated in sufficient quantities to permit further characterization. The partial amino acid analyses of each of the three forms were similar. They had commonextinction coefficients, E1(1)% cm A280, of 4.9 and A280/A290 ratio of about 1.36.

Immunoglobulin IgG1 metabolism in new born calves.

The half-life of IgG1 immunoglobulin was measured in six neonatal calves following a meal of iodine-125 labeled IgG1 in colostrum derived from their dams. The half-life as measured by the decrease in plasma concentration of IgG1 was 19.9 +/- 1.9 days. However, the half-life as measured by the disappearance of [iodine-125]IgG1 from the plasma was 11.5 +/- .6 days. The latter value is closer to the true half-life because it is not affected by endogenous production of IgG1 by the tissues of the young calf. A decrease in the specific activity of plasma [iodine-125]IgG1 with time representing the body "pool" of IgG1 (half-life 25.8 +/- 6.1 days) suggests that the calf from birth to about 20 days of age is capable of synthesizing a significant amount of IgG1 immunoglobulin.

Kinetic analysis of the binding of immunoglobulins IgG1 and IgG2 to bovine mammary cells.

Previous reports were confirmed that specific binding sites exist on bovine mammary cells near parturition presumably involved in the transfer of immunoglobulins IgG1 and IgG2 across the mammary gland at the time of colostrum formation. Determination of the kinetic parameters of these binding sites using 125I-labeled IgG1 and IgG2 immunoglobulins indicated the presence of sites with association constants (Ka) of about 5 - 10(8)--10 - 10(8)M-1 for both subclasses during normal lactation with about 9000 and 3000 sites per cell for each, respectively. The number of IgG1 sites tended to increase as the time of parturition approached. In addition, a new group of sites numbering about 5000 per cell with very strong binding for IgG1 (Ka about 45 - 10(8)M-1) appeared on the cells about a week before parturition. The numbers and affinity of the IgG1 and IgG2 binding sites bear a relationship to the approximate 7 : 1 ratio of these immunglobulin subclasses found in colostrum and normal milk and to the time of maximum colostrum formation. The results support the premise that a highly selective transport mechanism exists in the bovine mammary epithelial cell for the transfer of IgG1 and IgG2 immunoglobulins from blood to the lacteal secretions.

Production and turnover of IgG1 and IgG2 immunoglobulins in the bovine around parturition.

Production rates (entry rate into blood plasma) and other metabolic parameters for the IgG1 and IgG2 subclasses of immunoglobulins in mammary secretions (ratio of about 7 to 1) were determined in cows around the time of parturition by both single-injection and continuous-infusion isotope-dilution techniques. Four cows were given a single dose of 150 to 200 muCi of iodine-125 labeled IgG1 and 100 to 250 muCi of iodine-131 labeled IgG2 at 2 to 1 wk before parturition. Four cows, including two of the above, were infused continuously with constant amounts of the labeled immunoglobulins starting at 11 to 4 days before parturition. All cows were maintained until 4 to 6 days after parturition for monitoring the specific activities of iodine-125 labeled IgG1 and iodine-131 labeled IgG2 in the plasma and mammary secretions. Maximum entry rates of IgG1 and IgG2 were between 3 and 1 day prepartum with means of 125 and 60 g/500 kg body weight per day. The exchangeable pool means for IgG1 and IgG2 were 619 and 643 g/500 kg body weight, and both immunoglobulins were divided almost equally between the intra- and extravascular pools. A greatly increased production and a shortened half-life or greater turnover for plasma IgG1 occurs around the time of parturition which can account for the large accumulation of IgG1 in colostrum.

Comparative production of beta-lactoglobulin and orotic acid with lactose in bovine mammary cell cultures: effects of cell density and constituent inhibition.

Abilities to accumulate beta-lactoglobulin and ortic acid were compared to lactose in dispersed cell cultures of lactating bovine mammary tissue. The inverse of the amount accumulated of each milk constituent at a given time in the culture medium was a linear function of the inverse of the cell density. The amount of lactose had no effect on its own subsequent accumulation, but added orotic acid and beta-lactoglobulin inhibited their own production. The accumulation of certain milk constituents in the culture medium is a factor in the expression and loss of normal function in the in vitro cultures which may be related to the observed effects of milk accumulation in vivo on the rate of milk synthesis and mammary involution.

Effect of cell density on lactose synthesis in bovine mammary cell cultures.

The ability to synthesize lactose was studied in despersed cell cultures of lactating bovine mammary tissue under conditions of varying cell density, time, glucose, and lactose concentrations. The observable rate of lactose synthesis at a given time in an adequate medium is dependent on individual animal, length of time in culture, and density of cells. Rate of loss with time in the ability to synthesize lactose followed first order kinetics (half-time 11 h). Rate of synthesis per cell at a given time was an inverse function of the cell density following a linear relationship expressed by plotting the inverse of the amount accumulated versus the inverse of the cell number. A cell density for such experimental studies of about 2.5 times 10-6 cells per ml was ideal. The medium concentration of lactose and, above a minimum, that of glucose did not affect production of lactose.