Multi-parametric flow cytometric and genetic investigation of the peripheral B cell compartment in human type 1 diabetes.

The appearance of circulating islet-specific autoantibodies before disease diagnosis is a hallmark of human type 1 diabetes (T1D), and suggests a role for B cells in the pathogenesis of the disease. Alterations in the peripheral B cell compartment have been reported in T1D patients; however, to date, such studies have produced conflicting results and have been limited by sample size. In this study, we have performed a detailed characterization of the B cell compartment in T1D patients (n = 45) and healthy controls (n = 46), and assessed the secretion of the anti-inflammatory cytokine interleukin (IL)-10 in purified B cells from the same donors. Overall, we found no evidence for a profound alteration of the B cell compartment or in the production of IL-10 in peripheral blood of T1D patients. We also investigated age-related changes in peripheral B cell subsets and confirmed the sharp decrease with age of transitional CD19(+) CD27(-) CD24(hi) CD38(hi) B cells, a subset that has recently been ascribed a putative regulatory function. Genetic analysis of the B cell compartment revealed evidence for association of the IL2-IL21 T1D locus with IL-10 production by both memory B cells (P = 6·4 × 10(-4) ) and islet-specific CD4(+) T cells (P = 2·9 × 10(-3) ). In contrast to previous reports, we found no evidence for an alteration of the B cell compartment in healthy individuals homozygous for the non-synonymous PTPN22 Trp(620) T1D risk allele (rs2476601; Arg(620) Trp). The IL2-IL21 association we have identified, if confirmed, suggests a novel role for B cells in T1D pathogenesis through the production of IL-10, and reinforces the importance of IL-10 production by autoreactive CD4(+) T cells.

Outcome of functioning filtering blebs after pars plana vitrectomy.

BACKGROUND AND OBJECTIVE: To determine the status of filtering bleb function following pars plana vitrectomy. PATIENTS AND METHODS: The authors retrospectively reviewed patients with functioning filtering blebs undergoing pars plana vitrectomy. RESULTS: Twenty-three eyes with functioning filtering blebs underwent pars plana vitrectomy. Postoperatively, 7/23 (30 percent) of the eyes had moderate (5 to 20 mm Hg) intraocular pressure (IOP), 8/23 of the eyes had IOP persistently greater than 20 mm Hg, and 7/23 of the eyes had IOP of less than 5 mm Hg. One of 7 eyes that underwent vitrectomy within 6 weeks after trabeculectomy maintained bleb function, whereas 6 of 16 eyes that underwent trabeculectomy 6 weeks or later maintained bleb function. Loss of bleb function occurred in the early postoperative period in the majority of the patients. Prior antimetabolite therapy was not associated with preservation of bleb function. CONCLUSION: There is a substantial risk of bleb failure following vitrectomy, which is in part related to the often severe nature of the diseases requiring vitreoretinal surgery.

Endophthalmitis after penetrating trauma. Risk factors and visual acuity outcomes.

PURPOSE: To identify clinical characteristics that were associated with an increased incidence of endophthalmitis in eyes with penetrating ocular trauma. METHODS: In part 1, a retrospective analysis was performed on 258 consecutive patients with penetrating ocular trauma presenting to the Bascom Palmer Eye Institute between October 1987 and January 1991. In part 2 of the study, 28 consecutive patients with culture-proven endophthalmitis were identified from the Clinical Microbiology Registry from April 1987 through September 1987 and February 1991 through August 1993. Clinical variables were evaluated in each part for association with an increased risk of endophthalmitis. RESULTS: In part 1 of the study, endophthalmitis developed in 13 (5%) of the 258 patients. Endophthalmitis did not occur in eyes that had blunt injury. In those eyes with a lacerating injury, there was an increased relative risk of infection in eyes with disruption of the crystalline lens. This risk factor was found statistically significant by univariate and multivariate analysis. In part 2 of the study, lens disruption was present in 24 (86%) of 28 patients with culture-proven endophthalmitis. Of the 41 patients with infection from part I and part II, 22 (54%) achieved visual acuity of 20/ 400 or greater. Endophthalmitis caused by coagulase-negative staphylococci had the best visual outcome, with 7 (64%) of 11 patients obtaining visual acuity of 20/ 400 or greater. CONCLUSION: Lens disruption in eyes with penetrating trauma is a significant risk factor for the development of endophthalmitis. The prognosis for useful vision in eyes with posttraumatic endophthalmitis is best when infection is caused by less virulent organisms.

Acute retinal necrosis caused by reactivation of herpes simplex virus type 2.

Acute retinal necrosis is a severe form of necrotizing retinitis. Acute retinal necrosis has been demonstrated to be caused by varicella-zoster virus and herpes simplex virus type 1. We treated three patients with acute retinal necrosis apparently caused by recrudescence of latent herpes simplex virus type 2. Primary viral infection was probably congenital, with documented perinatal herpes simplex virus type 2 infection in two patients. Bilateral chorioretinal scars were present in two patients, neither of whom had a history of ocular herpetic infection, suggesting that earlier subclinical chorioretinitis had occurred. In each case, periocular trauma preceded the development of retinitis by two to three weeks. These cases are evidently caused by trauma-induced reactivation of latent virus rather than the onset of a primary infection.

The evaluation and management of corneal lacerations.

Corneal lacerations represent a significant portion of ocular trauma. Effective management of this type of injury involves a thorough evaluation to assess the severity of the injury and the development of a logical management plan. Minor corneal trauma may be handled on an out-patient basis with the use of contact lenses and tissue adhesives. Severe injuries generally require admission and surgical intervention. Utilizing kerato-refractive principles and new suture techniques, post-operative astigmatism can be minimized at the time of primary closure.

Antibodies introduced into living cells with liposomes localize specifically and inhibit specific intracellular processes.

We have developed a system for efficiently packaging antibodies and other macromolecules into liposomes and then delivering the encapsulated molecules into living cells through liposome-cell fusion. Fusion is very efficient, and all cells can be demonstrated to contain liposome-delivered antibodies by staining with a fluorescent second antibody. Using lupus antibodies directed against small nuclear ribonucleoprotein components of the cell, we were able to demonstrate strong nuclear localization, while control antibodies showed a general diffuse distribution throughout the cell. Lupus antibodies directed against ribosomes, on the other hand, strongly localized in the nucleolus and the cytoplasm with very little nucleoplasmic localization. Antitubulin antibodies predominantly localized in the cytoplasm. These results show that antibodies can survive liposome packaging and can retain their ability to recognize and bind to their specific antigens in the living cell. It also indicates that the nuclear envelope does not present a barrier to the liposome-introduced antibodies in Drosophila tissue culture cells. To determine if the antibodies were capable of interfering with cellular processes in vivo, we measured the effects of liposome-introduced antiribosome antibodies on translation and antitubulin antibodies on mitosis. In both cases, there was a significant inhibition suggesting that the antibodies can be used to interfere with specific functions at specific times in vivo.

Inherited deficiency of the Mac-1, LFA-1, p150,95 glycoprotein family and its molecular basis.

Leukocyte surface glycoproteins that share a common beta subunit have been found to be congenitally deficient in three unrelated patients with recurring bacterial infection. The glycoproteins, Mac-1, LFA-1, and p150,95, have the subunit compositions alpha M beta, alpha L beta, and alpha X beta, respectively. Using subunit-specific monoclonal antibodies, both the alpha M and beta subunits of Mac-1, the alpha L and beta subunits of LFA-1, and at the least the beta subunit of p150,95, were found to be deficient at the cell surface by the techniques of immunofluorescence flow cytometry, radioimmunoassay, and immunoprecipitation. A latent pool of Mac-1 that can be expressed on granulocyte surfaces in response to secretory stimuli, such as f-Met-Leu-Phe, was also lacking in patients. Deficiency was found on all leukocytes tested, including granulocytes, monocytes, and T and B lymphocytes. Quantitation by immunofluorescence cytometry of subunits on granulocytes from parents of these patients and of a fourth deceased patient showed approximately half-normal surface expression, and, together with data on other siblings and a family with an affected father and children, demonstrate autosomal recessive inheritance. Deficiency appears to be quantitative rather than qualitative, with two patients expressing approximately 0.5% and one patient approximately 5% of normal amounts. The latter patient had alpha beta complexes on the cell surface detectable by immunoprecipitation. Biosynthesis experiments showed the presence of normal amounts of alpha'L intracellular precursor in lymphoid lines of all three patients. Together with surface deficiency of three molecules that share a common beta subunit but have differing alpha subunits, this suggests the primary deficiency is of the beta subunit. The lack of maturation of alpha'L to alpha L and the deficiency of the alpha subunits at the cell surface and in latent pools suggests that association with the beta subunit is required for alpha subunit processing and transport to the cell surface or to latent pools. The molecular basis of this disease is discussed in light of adhesion-related functional abnormalities in patients' leukocytes and the blockade of similar functions in healthy cells by monoclonal antibodies.

Simian virus 40 (SV40)-specific isoelectric point-4.7--94,000-Mr membrane glycoprotein: major peptide homology exhibited with the nuclear and membrane-associated 94,000-Mr SV40 T-antigen in hamsters.

Tryptic peptide maps of electrophoretically purified 94,000-molecular weight (relative) (Mr) nuclear and membrane-associated simian virus 40 (SV40) T-antigens, TN and TM, respectively, were compared to those of the SV40-specific isoelectric point (pI)- 4.7--94,000-Mr plasma membrane component reactive with anti-T-sera from Syrian golden hamsters. Bidimensional thin-layer electrophoresis and chromatography of TN labeled with 125I revealed about 27 tryptic peptides. A similar number of peptides was identified for TM and the pI-4.7--94,000-Mr component. A peptide homology between TN and TM or TN and the pI-4.7-94,000-Mr protein exists and indicates that the previously described pI-4.7--94,000-Mr membrane component represents TM. Only 4 of 27 peptides were labeled when TM was subjected to lactoperoxidase-catalyzed radioiodination from the outer surface of the plasma membrane. One of these TM peptides was metabolically labeled with [14C]glucosamine. The data indicate that TM is partially exposed on the cell surface and represents a glycosylated form of TN. Closely associated with TM is a pI-4.5--55,000-Mr membrane component. This component does not exhibit significant peptide homology with the 94,000-Mr SV40 protein and, therefore, appears to be coded for by the host cell genome.

Host cell-modified T-antigen in membranes of simian virus 40-transformed hamster cells.

Highly purified plasma membranes of simian virus 40 (SV40)-transformed hamster and mouse cells were subjected to indirect immunoprecipitation and bidimensional isoelectric focusing-immunoelectrophoresis with high-titer (greater than or equal to 512) sera against SV40 T-antigen. An SV40-specific protein of approximately 100,000 daltons and pH-4.7 isoelectric point cross-reacted immunologically with T-antigen, which indicated the presence of a T-antigen species. However, this protein appeared to be host cell modified because of its low isoelectric point and its reactivity with heterologous antisera containing antibodies specific for neuraminidase- and trypsin-sensitive carbohydrate and/or peptide moieties lacking nuclear T-antigen. Another protein specific for the membranes of SV40-transformed cells had a molecular mass near 60,000 daltons and an isoelectric point at pH 4.5 and appeared closely associated with membrane T-antigen. It coprecipitated with membrane T-antigen upon direct immunoprecipitation with anti-T serum. However, when this protein was dissociated from membrane T-antigen by isoelectric focusing in the presence of Triton X-100 and urea, its reactivity with anti-T serum was lost. This suggested that the protein was not encoded in the SV40 genome.

Pili multigemini. Report of a case in association with cleidocranial dysostosis.

A patient with cleidocranial dysostosis developed extensive pili multigemini over the heavily bearded chin and cheek areas. Histological examination of serial sections revealed complicated follicular structures forming from two to as many eight hair shafts. Each hair is formed by a single branch of dermal papilla which is surrounded by all layers present in a normal follicle except for the outer root sheath cells. The outer root sheath surrounds the entire follicle. Irregularities in configuration of the hairs, longitudinal grooving and areas of bifurcation and re-adhesion of the hair shafts are demonstrated.

Inactivation of Salmonellae in Autoclaved Ground Beef Exposed to Constantly Rising Temperatures 1.

Inactivation of a composite of five serotypes of salmonellae was monitored in autoclaved ground beef exposed to constantly rising temperatures increased at rates similar to those used in beef cookery. Rising temperature rates of 6.0 C/h, 8.5 C/h and 12.5 C/h and constant temperatures of 55, 57, 61 and 63 C were examined. Survival of Salmonella typhimurium TM-1 was compared to survival of the composite. D and z values were compared for constant and rising temperature rates. The D50 C for constant temperature data was 30.2 min, and the D50 C for changing temperature data was 78.6 min (6.0 C/h), 82.4 min (8.5 C/h), and 49.8 min (12.5 C/h). Neither serotype nor heat treatment of ground beef had a major influence on apparent heat resistance of salmonellae. A comparison of these results to previous rising temperature work with Clostridium perfringens suggested that controlling C. perfringens will result in control of salmonellae. On the basis of these results, the July 18, 1978, USDA processing ruling appears adequate to control salmonellae in precooked beef roasts.

Simian virus 40-specific proteins in the membranes of simian virus 40-transformed hamster and mouse cells.

Membranes of simian virus 40-transformed hamster lymphocytes and phagocytes, as well as of transformed mouse fibroblasts, contain two classes of antigenic virus-specific protein. The isoelectric points of these proteins, as defined by isoelectric focusing/immune electrophoresis are at pH 4.5 and 4.7. The molecular weights of the pI 4.5 and pI 4.7 components, determined by isoelectric focusing/dodecyl sulfate polyacrylamide electrophoresis, lie near 58,000 and 90,000-110,000, respectively. The pI 4.5 and pI 4.7 proteins are tentatively identified with the surface (transplantation) and U antigens, respectively.

Differences between the structural dynamics of plasma membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40 as revealed by laser Raman spectroscopy.

The Raman spectra of highly purified plasma membranes from SV40-transformed GD248 lymphocytes have been compared with the spectra of the membranes of normal cells over the spectral region 100 cm-1 to 3010 cm-1. Striking differences between the two membrane categories were observed in the thermal response of the CH-stretching and acoustical regions. Analysis of CH-stretching shows that the membranes of normal cells exhibit a thermal transition centered at 7 degrees and approximately 5 degrees wide. The membranes of GD248 cells, in contrast, show a lipid transition centered at -5 degrees and 12-18 degrees wide. Analysis of the acoustical region yields equivalent results. The membrane proteins of normal membranes undergo a large thermotropic transition, starting at 39 degrees (sample temperatures), whereas this transition begins at 23 degrees with GD248 plasma membranes. The results suggest the possibility that SV40-specific membrane proteins may modify the collective thermotropic behavior of both normal membrane proteins and membrane lipids.

Antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40.

Highly purified plasma membranes from hamster lymphocytes transformed by simian virus 40 (GD 248) were compared with the membranes of normal cells by crossed immune electrophoresis, crossed-line immune electrophoresis, and bidimensional isoelectric focusing-immune electrophoresis. Antiserum raised by inoculation of guinea pigs with GD 248 membranes was used as serologic reagent, either directly or after absorption with membranes from normal cells. Bidimensional immune electrophoresis reveals the presence in the plasma membranes of GD 248 cells of at least three antigens not detectable in the membranes from the normal cell population. At least two of these are also present in the mitochondrial membranes of GD 248 cells, but none could be detected in membranes of embryonic fibroblasts. Bidimensional isoelectric focusing-immune electrophoresis indicates that the distinctive antigens of the GD 248 membranes are glycoproteins.