Nonspecific signals for B-cell localization and activation.

Virgin, inactive mammary gland autografted to the anterior chamber of the rabbit eye remains free of lymphoid cells. Activation of the ectopic gland by systemic injection of chorionic gonadotropin results in maturation of the gland and milk production, accompanied by the immirgration of lymphocytes and their activati-n to Ig formation, predominantly of the IgA class. In the presence of antigen-induced intraocular inflammation, the activated gland is able to influence the Ig class of B cells in the neighboring ocular tissues. These data suggest that even nonlymphoid tissues may elaborate lymphocyte-homing and polyclonal B-cell activating factors which function independently of specific antigen.

Plasticity of heart rate signalling and complexity with exercise training in obese individuals with and without type 2 diabetes.

OBJECTIVE: To examine the responsiveness of cardiac autonomic function and baroreflex sensitivity (BRS) to exercise training in obese individuals without (OB) and with type 2 diabetes (ObT2D). DESIGN: Subjects were tested in the supine position and in response to a sympathetic challenge before and after a 16-week aerobic training program. All testing was conducted in the morning following a 12-h fast. SUBJECTS: A total of 34 OB and 22 ObT2D men and women (40-60 years of age) were studied. MEASUREMENTS: Heart rate variability (HRV) was measured at rest via continuous ECG (spectral analysis with the autoregressive approach) and in response to upright tilt. The dynamics of heart rate complexity were analyzed with sample entropy and Lempel-Ziv entropy, and BRS was determined via the sequence technique. Subjects were aerobically trained 4 times per week for 30-45 min for 16 weeks. RESULTS: Resting HR decreased and total power (lnTP, ms(2)) of HRV increased in response to exercise training (P<0.05). High frequency power (lnHF) increased in OB subjects but not in OBT2D, and no changes occurred in ln low frequency/HF power with training. Upright tilt decreased lnTP and lnHF and increased LF/HF (P<0.01) but there were no group differences in the magnitude of these changes nor were they altered with training in either group. Tilt also decreased complexity (sample entropy and Lempel-Ziv entropy; P<0.001), but there was no group or training effect on complexity. BRS decreased with upright tilt (P<0.01) but did not change with training. Compared to OB subjects the ObT2D had less tilt-induced changes in BRS. CONCLUSION: Exercise training improved HRV and parasympathetic modulation (lnHF) in OB subjects but not in ObT2D, indicating plasticity in the autonomic nervous system in response to this weight-neutral exercise program only in the absence of diabetes. HR complexity and BRS were not altered by 16 weeks of training in either OB or ObT2D individuals.

Reversal of T-cell unresponsiveness by T-cell-conditioned medium in retrovirus MAV-2-O-induced immunosuppression in chickens.

Polyclonal activation of T-cells with concanavalin A has been used as an in vitro test system to study immunosuppression induced by the avian retrovirus MAV-2-O. The mitogenic responsiveness of peripheral blood lymphocytes from infected chickens was only weakly suppressed, that of spleen lymphocytes was highly suppressed. Addition of conditioned media rich in T-cell growth factor (TCGF) activity to cultures of infected birds resulted in a reconstitution of the suppressed mitogenic responsiveness up to the level found in lymphocyte cultures from normal chickens. This indicates that the defect of the observed immunopathology is not at the level of responding T-cells. Measurements of TCGF production always showed reduced TCGF levels in the suppressed cultures suggesting that there is a defect at the TCGF production level. This is further supported by the failure to reconstitute the suppressed mitogenic response with normal macrophages, which are involved in activation of TCGF producer T-cells.

Identification of acyl phosphatidylglycerol as a minor phospholipid of Pseudomonas BAL-31.

Compound X, a minor phospholipid of Pseudomonas BAL-31 and bacteriophage PM2, has been identified as X-3-phosphatidyl-1'-(3'-acyl)-glycerol, or acyl phosphatidylglycerol. The water-soluble product obtained by mild alkaline hydrolysis showed the same RF value as that of glycerophosphoryl-glycerol. The chemical analysis gave the ratio 1 : 3 : 2 for phosphate-acyl ester-glycerol. The position of the third acyl group was determined by nuclear magnetic resonance techniques.

Small angle x-ray scattering studies on adenovirus type 2 hexon.

Adenovirus type 2 hexons have been studied in solution by small angle X-ray scattering, and the following molecular parameters determined: radius of gyration (Rg) = 4.9 nm, molecular weight (M) = 310.000, invariant volume (Vinv) = 630 mn3, maximal distance (Dmax) = 14.5--15.5 nm. A diffraction pattern was obtained up to an angular increment of h = 2.5 nm-1. Various models for the hexon have been explored by calculating the diffraction pattern from the Debye formula for 1200 spheres arranged to define the scattering volume of each model. Models were first built according to electron micrographic results. Later, preliminary results of a crystallographic study were used for model building. The experimental pattern and the pattern resulting from the model determined by crystallographic methods were compared and showed good agreement.

A novel method to improve immunofluorescence in histological specimens.

A new approach to immunofluorescent labeling of sectioned tissue is described. Small tissue fragments which have been fixed and rendered permeable are labeled with immune reagents by either the direct or indirect method, prior to embedding in glycol methacrylate (2-hydroxyethyl methacrylate) at 4 degrees. In this way, the well-known superiority of plastic embedding over paraffin embedding or cryo-sections can be used to compare cellular structure in tissues with that seen in cultured cells.

Expression and secretion of malarial parasite beta-tubulin in Bacillus brevis.

Microtubule inhibitors are active against the human malarial parasite Plasmodium falciparum, but whether these drugs actually interact with parasite tubulins is not known. It has not previously been possible to produce mg quantities of isolated, soluble tubulin subunits for drug-binding experiments. A cDNA encoding P falciparum beta-tubulin was expressed and the protein secreted in Bacillus brevis. With the addition of EGTA to the culture medium, which increases shedding of proteins from the cell surface, up to 2 mg/l recombinant beta-tubulin was obtained in supernatants. It is not clear why B brevis is able to secrete this normally cytoplasmic protein, but the secretion levels of recombinant proteins may be related to the net charge of the first few residues of the mature polypeptide.

Immunohistochemistry on semi-thin sections of hydroxypropyl methacrylate embedded tissues.

Hydroxypropyl methacrylate formulations for embedding hard or soft tissues are described. Dehydration, infiltration and embedding are carried out at 0-4 degrees C. Semi-thin serial sections may be stained by various techniques, including immunohistochemical and enzyme histochemical procedures. Lymphoid tissues and the detection of surface antigens may present some special problems, however, and these are discussed.

Display and analysis of cell size distributions with a Coulter Counter interfaced to an Apple II microcomputer.

A model ZBI Coulter Counter was interfaced to an Apple II microcomputer via analog and digital interfaces. It was possible to obtain a graphic display of cell size versus channel number (1000 channels), integration of selected channels, plots, listings, and storage of the data on floppy disk, using a convenient menu selection enabling operators unfamiliar with computer technology to master the system quickly. Examples are presented of calibration of the Coulter Counter and its use for rapid and precise analysis of E rosettes.

Localization and stage specific phosphorylation of Plasmodium falciparum phosphoproteins during the intraerythrocytic cycle.

Fifty-nine Plasmodium falciparum specific phosphoproteins with molecular weights between 15,000 and 192,000 were analyzed by SDS-PAGE and two-dimensional gel electrophoresis. 40 phosphoproteins were identified by [gamma-32P]ATP labeling of cell lysates, 19 by [32P]orthophosphate labeling of parasitic cultures in vivo. Changes in the phosphorylation pattern during the infectious erythrocytic cycle were determined for all proteins. In parallel, cell fractionation studies were done to follow up possible changes in the cellular distribution of these proteins. Several phosphoproteins are associated with the membrane fraction of infected erythrocytes. One pair of proteins of approx. 88 kDa and a pI of about 5.0 was further characterized. Both proteins are located in the parasitic fractions as well as in the membrane of infected erythrocytes during the entire cycle. Phosphorylation of these proteins, however, is restricted to the trophozoite and schizont stages. Peptide mapping studies demonstrated that both proteins are identical with the exception of minor modifications which are probably not the result of differences in phosphorylation.

Plasmodium falciparum calcium-dependent protein kinase phosphorylates proteins of the host erythrocytic membrane.

The unusual Ca(2+)-dependent protein kinase from Plasmodium falciparum (PfCPK) [1], whose gene structure and expression in bacteria have been reported [1], was purified to homogeneity. The purified recombinant kinase has a native molecular mass of 62,000, is activated by Ca2+ (K0.5 = 15 microM) in the presence of Mg2+ or Mn2+, and can associate with 45Ca2+. The activation by Ca2+ could be partially replaced by Mn2+, but not by Zn2+ or Mg2+. PfCPK preferentially phosphorylated casein and histone H1. The Km and Vmax for Mg2+ ATP were 26 microM and 70 nmol min-1 mg-1, respectively, with casein as substrate; and 34 microM and 143 nmol min-1 mg-1, respectively, with histone H1 as substrate. The kinase undergoes autophosphorylation on both serine and threonine residues. Calmodulin antagonists (calmidazolium, trifluoperazine, N-[6-aminohexyl]-5-chloro-1-napthalene-sulfonamide, and ophiobolin A) could inhibit the kinase activation, but much higher concentrations of the antagonists are needed than was required to inhibit calmodulin-mediated effects. PfCPK preferentially phosphorylates proteins of the host erythrocytic membrane in vitro but phosphorylates parasitic proteins only to a minor extent. The selectivity of the phosphorylation may be partially controlled by phosphatidylserine which is bound to some of the erythrocytic membrane proteins. Using a rabbit polyclonal antiserum against the recombinant protein, the kinase was found to be mainly expressed in the ring and schizont stages, and mainly localized in the parasitic membrane-organelle fraction and partially localized on the erythrocytic membrane.

Plasmodium falciparum protein kinase 5 and the malarial nuclear division cycles.

In the course of our studies on cell cycle regulation mechanisms of Plasmodium falciparum, we investigated expression pattern, kinase activity, and localization of PfPK5, a putative malarial member of the family of cyclin-dependent protein kinase (cdks). The kinase was immunoprecipitated from parasites of selected stages and from parasites blocked with the cell-cycle inhibitor aphidicolin. An elevated kinase activity of PfPK5 from aphidicolin-blocked cells suggested that the enzyme might be implicated in the regulation of the parasite's S-phase. To further investigate this hypothetical function, parasite cultures were treated with the specific cdk inhibitors flavopiridol and olomoucine, which act on PfPK5 in vitro at similar concentrations as on other cdks. When applied during the nuclear division cycles of the parasite, both drugs markedly inhibited the DNA synthesis, as predicted from our proposition that PfPK5 is necessary to activate or maintain the parasite S-phase. Immunolocalization studies provide further evidence for this potential role of PfPK5.

A Plasmodium falciparum protein kinase with two unusually large kinase inserts.

A protein kinase gene (PfPK1) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases. The gene, which contains one intron, encodes a protein with a predicted length of 909 amino acids. The predicted protein contains all the conserved sequences characteristic of a protein kinase catalytic domain. These sequences are discontinuous, however, since they are separated by two large kinase inserts with 178 and 330 amino acids in size. Specific antisera were raised against recombinant fragments of the protein and a PfPK1-specific peptide. Using one of these antibodies, a functional protein kinase was precipitated from malarial lysates and this kinase recognized casein as an exogenous substrate. PfPK1 was expressed in a stage-specific fashion and also had a stage-specific cellular localization. During the intraerythrocytic life cycle, PfPK1 shifts from the parasite cytosol to the parasite membrane fraction. An unusual feature of PfPK1 is its electrophoretic mobility on SDS-PAGE. Whereas the predicted protein size is about 100 kDa, the apparent size is about 70 kDa. There are no indications for RNA processing and we could exclude proteolytic processing as an explanation.

Two major phosphoproteins of Plasmodium falciparum are heat shock proteins.

Two major phosphoproteins of Plasmodium falciparum could be identified by partial amino acid sequencing as the plasmodial members of the hsp 70 heat shock protein family, Pfhsp and Pfgrp. According to phosphoamino acid analyses of Pfhsp and Pfgrp isolated from [32P]orthophosphate-labeled malarial cultures, both proteins were phosphorylated in Ser and Thr. While Pfhsp contains higher amounts of labeled phosphoserine, Pfgrp contains higher amounts of phosphothreonine. Phosphorylation of both proteins increased throughout the entire erythrocytic growth cycle. At the trophozoite and schizont stages Pfhsp and Pfgrp are the most prominent phosphoproteins of Plasmodium falciparum. Using multiply redundant oligonucleotides directed against the N-terminus of Pfgrp we cloned and sequenced the entire Pfgrp gene. The gene encodes a product with a predicted length of 652 amino acids. The deduced amino acid sequence has identities of 65.5% and 65.0% to the human and rat grp78 proteins, respectively. Pfgrp possesses a classical N-terminal leader sequence. The published grp78 related gene sequences of Plasmodium falciparum are all fragments of the same plasmodial gene.

Conjunctival-associated lymphoid tissue: evidence for a role in the secretory immune system.

A specialized lymphoid tissue in rabbit conjunctiva was studied by various histologic and immunologic techniques and compared with similar structures along other mucosal surfaces. The flattened conjunctival lymphoepithelium overlying the lymphoid follicles was devoid of goblet cells. This lack of goblet cells is characteristic of epithelium overlying similar lymphoid collections in gut and bronchus. The lymphoid follicles demonstrated neither intra- nor extracellular immunoglobulin, and the lymphocytes in these follicles were composed of B-cells and T-cells, when studied by various immunologic techniques. A high proportion of these lymphocytes showed surface immunoglobulin A (IgA), and a high proportion of IgA precursors were determined by pokeweed mitogen (PWM) stimulation in 4-day cultures. The morphologic and immunologic results are similar to those obtained from gut and bronchus, tissues known to disseminate lymphoid cells to other mucosal sites already committed to antigen and IgA isotype. It is speculated that conjunctival associated lymphoid tissue of rabbit is part of a generalized system of secretory immunity capable of sampling conjunctival applied antigen, and then disseminating cells committed to IgA antibody production to other mucosal sites.

Mucus-stimulating factor in tears.

Mechanisms responsible for regulation of tear film mucus are poorly understood. Humoral factors responsible for stimulation of mucus secretion can be studied in vitro by using the free-swimming urn cell, a normal component of the coelomic fluid of the marine invertebrate Sipunculus nudus. With this system, a tear mucus-stimulating factor was found in normal human tears but was markedly decreased in patients with dry eye syndromes. It is suggested that a mucus-stimulating factor exists in normal human tears and that a decrease in this substance may be instrumental in the pathophysiology of certain dry eye syndromes.

Secretory immune system of rabbit ocular adnexa.

A distinct system of immunity in a variety of animals is located subjacent to epithelial surfaces and is typified by the predominance of immunoglobulin A (IgA) and secretory component (SC) in various external secretions, including tears. The present study examined normal rabbit lacrimal gland, conjunctiva, and cornea for the presence of immunoglobulin and SC. IgA-staining plasma cells predominated within lacrimal gland and conjunctival stroma, and SC was found in the epithelial cells of both these tissues but not within corneal epithelium. These observations are consistent with findings for other secretory sites in both rabbits and humans and establish lacrimal gland and conjunctiva as integral parts of the rabbit secretory immune system.

Lacrimal gland-derived lymphocyte proliferation potentiating factor.

PURPOSE: To determine whether lacrimal gland acinar cells are involved in modulating local immune response; to isolate and characterize a lacrimal gland factor that exerts biological activities on T lymphocytes. METHODS: A protein factor has been purified from lacrimal gland extracts by a combination of ion exchange and gel-filtration chromatography. This factor has the capacity to enhance proliferation of T lymphocytes upon stimulation with a mitogen or an antigen. We have, therefore, called this substance lacrimal gland-derived lymphocyte proliferation potentiating factor (LG-F). RESULTS: Lacrimal gland-derived lymphocyte proliferation potentiating factor has a molecular weight of approximately 65,000 daltons as determined by gel-filtration and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. T cells demonstrate greater proliferation when cultured with high concentrations of concanavalin A (Con A) in the presence of LG-F, as compared with culture without addition of LG-F. This enhancing effect of LG-F may be mediated by IL-2, because the final cell count correlates with the levels of IL-2 secreted by LG-F-activated cells. Lacrimal gland-derived lymphocyte proliferation potentiating factor is nonmitogenic for T cells, but its potentiating effect is antigen-dependent. Dual stimulation of OA-primed T cells with both OA and LG-F results in greater proliferative activity, in contrast to culture with OA alone. CONCLUSIONS: The results suggest that lacrimal gland cells may interact with the immune system by elaborating nonspecific factors that modulate lymphocyte proliferation and augment lymphokine production. The presence of such a factor in the lacrimal gland may prove to be of importance in the generation of local immune responses.

Secretory component of IgA: a marker for differentiation of ocular epithelium.

Secretory component (SC) was studied by indirect immunofluorescence of the ocular surface epithelium. We find that conjunctival epithelium produces this component, and that it is absent in the corneal epithelium. Conjunctival epithelium loses its SC staining within 1 to 2 days as it grows over a denuded corneal stroma. This implies a very rapid turn-off of the SC gene and rapid export of the gene product previously formed. However, conjunctival flap epithelium does not change its characteristic structure or functions. Vascularization of corneas resurfaced by conjunctival epithelium usually leads to a rapid reversal of both morphological and biochemical characteristics in the surface epithelium, as judged by the reappearance of goblet cells and positive staining for SC. Vascularization of normal corneas leaves the epithelium unchanged, so that neither goblet cells nor SC appear. Thus metaplasia of conjunctival to corneal epithelium is incomplete, permitting reversion to type under certain conditions.

The age-dependent size distribution of chicken peripheral blood cells analyzed by a novel method utilizing a Coulter counter.

In a dilute solution of Tris-buffered ammonium chloride (TAC), chicken erythrocytes (RBC) swell but chicken white blood cells (WBC) do not. This allowed us to separate the peaks of RBC and wBC into widely separate channels using a Coulter Counter interfaced to a microcomputer to provide size distributions. Analyses of buffy coat by this technique were carried out on Brown Leghorn chickens from 1 to 21 weeks after hatching. The WBC population, which is chiefly composed of peripheral blood lymphocytes, increased in number from 2 to 7 weeks after hatching, but decreased in size continuously up to the end of the experiment at 21 weeks. The RBC count increased from 2 to 5 weeks after hatching and the size decreased up to 5 weeks after hatching, thereafter remaining constant.