SARS-COV-2 viral load in tears of patients with COVID-19 in the early symptomatic stages: comparison of two different tear sampling methods.

PURPOSE: To evaluate the presence of SARS-CoV-2 virus in tears of patients with COVID-19 in the early symptomatic stages and to compare two different sampling methods. MATERIALS AND METHOD: In this cross-sectional study, tears sampling was performed in COVID-19 patients admitted within the first 7 days of symptom onset. The samples were collected with both conjunctival swabs and Schirmer strips. Each specimen was analyzed via RT-PCR. The viral load was evaluated in terms of the cycle threshold value. Ocular and systemic symptoms and comorbidities of the patients were also recorded. RESULTS: Forty patients were included. The average time from the initiation of symptoms was 3.15 days. Unilateral conjunctivitis has been observed in 5% of patients and foreign body sensation in 7.5% of patients. No viral RNA was detected in the tear samples of the patients with ocular findings. The positivity rate for SARS-CoV-2 in tears was 2.5% (n = 1). None of the samples collected by Schirmer test strips yielded positive polymerase chain reaction result for SARS-COV-2. The Ct value of the positive conjunctival swab was 36.03 and the nasopharyngeal Ct value of the same patient was 25.68. CONCLUSION: The SARS-CoV-2 viral shedding rate has been determined as 2.5% in the tears of early symptomatic stage COVID-19 patients. The viral load of the tears was lower than the naso-oropharynx. The conjunctival swab method is recommended in tear collection to evaluate the presence of SARS-CoV-2 by RT-PCR analysis in low viral load tears.

Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins.

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.

Apical endocytosis of ricin in MDCK cells is regulated by the cyclooxygenase pathway.

Addition of arachidonic acid or stimulation of arachidonic acid production by secretory phospholipase A2 selectively upregulated apical endocytosis of ricin in MDCK cells without affecting basolateral endocytosis. Electron microscopic studies revealed that MDCK cells treated with secretory phospholipase A2 and incubated with horseradish peroxidase had an increased number of normal appearing peroxidase-labeled endosomes and no sign of membrane ruffling. Moreover, inhibition of basal arachidonic acid release, either by decreasing the cytosolic phospholipase A(2) activity or the diacylglycerol lipase activity, reduced the rate of apical endocytosis. Furthermore, indomethacin, an inhibitor of the cyclooxygenase pathway, counteracted the stimulation of endocytosis seen with both secretory phospholipase A2 and arachidonic acid, suggesting that formation of eicosanoids such as prostaglandins could be essential for the regulation. This idea was supported by the finding that prostaglandin E2, the predominant prostaglandin formed in kidney, also upregulated ricin uptake. The regulatory effect of the cyclooxygenase pathway on apical endocytosis of ricin was found to be independent of protein kinases A and C, which are known to selectively control apical clathrin-independent endocytosis in polarized cells.

Effect of calmodulin antagonists on endocytosis and intracellular transport of ricin in polarized MDCK cells.

The effect of calmodulin antagonists on endocytosis, transcytosis, recycling, and transport to the Golgi apparatus from both the apical and the basolateral plasma membrane of polarized Madin-Darby canine kidney cells has been investigated by using the plant toxin ricin as a membrane marker. The calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) stimulated apical endocytosis of ricin, whereas basolateral endocytosis was unaffected. A stimulation of the apical uptake of the fluid-phase marker horseradish peroxidase by calmodulin antagonists was also found both by biochemical and by ultrastructural studies. Furthermore, W-7 reduced the recycling of ricin to the apical plasma membrane, whereas the recycling to the basolateral plasma membrane was not changed. Transport of ricin to the Golgi apparatus was also selectively affected by the calmodulin antagonist W-7. After basolateral endocytosis of ricin, transport to the Golgi apparatus was reduced, whereas after apical endocytosis the fraction of endocytosed ricin transport to the Golgi apparatus was increased. Transcytosis of ricin from the basolateral to the apical pole was increased in the presence of calmodulin antagonists, whereas these compounds did not have any significant effect on the apical to basolateral transcytosis. Thus, the results obtained indicate that calmodulin is involved in regulation of apical endocytosis and recycling as well as in transcytosis of ricin from the basolateral plasma membrane. Furthermore, the data suggest that calmodulin plays a role in regulation of ricin transport to the Golgi apparatus.

Phorbol myristate acetate selectively stimulates apical endocytosis via protein kinase C in polarized MDCK cells.

We have examined the regulation of endocytosis in polarized Madin-Darby canine kidney (MDCK) cells. Using quantitative electron microscopy and biochemical measurements, we found that incubation with the tumor promoter phorbol 12-myristate 13-acetate (TPA) stimulated endocytosis of horseradish peroxidase (HRP) and ricin four- to fivefold at the apical side in MDCK cells, whereas the uptake at the basolateral membrane was unaffected. The use of several protein kinase inhibitors and TPA analogues indicated that the stimulation of apical endocytosis was mediated via protein kinase C independently of protein kinase A. This stimulation occurred even when the clathrin-dependent pathway was inhibited by acidification of the cytosol, suggesting that the TPA-stimulated uptake was associated with a clathrin-independent mechanism. Moreover, we found that TPA also stimulated recycling of ricin to the apical domain. Ultrastructural analysis of MDCK cells preincubated with TPA revealed that neither the morphology nor the size of the endosomes was altered compared to control cells. Using morphometry, no marked change in the apical plasma membrane area was detected after incubation with TPA. These data indicate that the TPA-stimulated endocytosis involved neither ruffling nor formation of macropinosomes in MDCK cells. Finally, we found that TPA also selectively stimulated apical endocytosis in the human colon adenocarcinoma cell line (Caco-2). The data suggest that protein kinase C is involved in a strong stimulation of apical endocytosis and might participate in the regulation of membrane trafficking between the apical plasma membrane and apical endosomes in polarized epithelial cells.

Selective regulation of apical endocytosis in polarized Madin-Darby canine kidney cells by mastoparan and cAMP.

We here demonstrate that mastoparan, fluoride, forskolin, cholera toxin, and 8-bromoadenosine-3'-5'-cyclic monophosphate all selectively stimulate apical endocytosis of the protein toxin ricin without increasing the uptake at the basolateral side of polarized Madin-Darby canine kidney cells. Activation of adenylyl cyclase and an increased level of cAMP seem to increase ricin endocytosis by a clathrin-independent mechanism since stimulation of endocytosis by cholera toxin and 8-bromoadenosine-3'-5'-cyclic monophosphate occurred even when the clathrin-dependent pathway was blocked by low cytosolic pH. The data suggest that mastoparan stimulates apical endocytosis by interacting with heterotrimeric G proteins, and also this stimulation of endocytosis appears to be clathrin independent since the uptake of transferrin at the apical side was strongly inhibited by mastoparan after brefeldin A-induced missorting of the transferrin receptor to this pole of the cell. In addition, mastoparan stimulated apical endocytosis when clathrin-mediated endocytosis was blocked by acidification of the cytosol. Furthermore, we provide evidence for the existence of clathrin-independent endocytosis on both the apical and the basolateral surface of control Madin-Darby canine kidney cells. Our results suggest that endocytosis at the apical pole of epithelial cells can be regulated selectively by a physiological signal.

Effects of epidermal growth factor, fibroblast growth factor, retinoic acid and serum on anchorage-dependent and anchorage-independent growth of HRRT cells.

The effects of EGF, FGF, RA and serum on anchorage-dependent and anchorage-independent growth of HRRT cells were studied. The five different types of serum tested in the present work induced a dose dependent rise in anchorage-independent growth in aggregates. FCS, SBCS and RS also supported colony formation in soft agar, whereas BS and HS had no significant effect. EGF and FGF stimulated anchorage-dependent growth of HRRT cells in monolayers. The peptide growth factors were also found to induce phenotypic transformation of the nonneoplastic HRRT cells, as measured by anchorage-independent growth in soft agar as well as in aggregates. At equimolar concentrations EGF was much more effective than FGF. The stimulating effect of EGF and FGF on cell proliferation in the aggregate form was markedly inhibited by RA. Treatment of HRRT cells with the highest noncytotoxic concentration of RA, 2 x 10(-7) M, reduced the stimulating effect of both growth factors by about 60%.

Initiation of in vitro cell transformation by formaldehyde and acetaldehyde as measured by attachment-independent survival of cells in aggregates.

The ability of formaldehyde and acetaldehyde to initiate transformation of a rat kidney cell line has been studied using a newly developed two-stage in vitro cell transformation assay. The assay is based on measurements of attachment-independent survival of cells in aggregates. Short treatment with non-cytotoxic doses of formaldehyde and acetaldehyde did not affect survival of the cells in the aggregate assay system. However, when the aldehyde treatment was followed by exposure of the cells to the tumor promoters TPA and PDD, a considerable increase in the number of viable cells was observed. On a molar basis, formaldehyde was about 100 times more potent than acetaldehyde in initiation of cell transformation. The data showed that cells derived from aggregates of cultures treated with formaldehyde or acetaldehyde followed by exposure to TPA possessed a considerably higher ability to form colonies in soft agar than untreated control cells.

Establishment and characterization of a new cell line from a hereditary renal rat tumor.

A new cloned tissue culture cell line, HRRT, has been established from a hereditary renal rat tumor. Electron microscopic studies indicate that the cells are fibroblasts. The cell line has been maintained in monolayer cultures for more than 3 yr and multiplies with a population doubling time of 21 h. The HRRT cells were found to have a plating efficiency of 50% and were not able to grow at low serum concentrations; they did not multiply in suspension culture and were unable to form colonies in soft agar or to proliferate in the aggregate form. The HRRT cells did not form tumors in nude mice. The new cell line was adapted to grow in serum-free medium by decreasing the serum concentration gradually over a period of several months and by addition of sodium pyruvate, insulin, alanine, serine, ribose, thymidine, and uridine to the medium. In serum-free medium HRRT cells multiplied with a population doubling time of 42 h and were not able to form colonies in soft agar.

Early detection of breast cancer by leukocyte adherence inhibition assay.

The hematocytometer leukocyte adherence inhibition technique was used to study cell-mediated immune activity against breast carcinoma. In a group of 83 patients with untreated breast cancer in stage I, 74% showed a positive response, among 47 patients in stage II, 64% responded, while only 51% of the 37 patients in stages III and IV responded. Of 86 control persons, only two women showed a positive response. In a group of 296 women with benign breast disease, 21% showed a positive reaction against breast carcinoma antigen. The percentage of positive responses was higher than the average among women with risk factors such as: mother or sister with breast cancer, previous removal of benign breast lumps, microcalcifications, and increased intraductal epithelial proliferation found in the biopsies. Women with benign breast disease having two or more of the above risk factors were assigned to the high risk group. Of 49 women in this group, 47% had reactive leukocytes. Ninety-two women had only one risk factor, and 27% of those showed a positive reaction. Of the 155 women with none of the risk factors, only 10% had a positive reaction. The results suggest that leukocyte adherence inhibition test may be used to identify groups of women with an increased possibility of developing breast cancer.

Assay for initiators and promoters of carcinogenesis based on attachment-independent survival of cells in aggregates.

A cell line (HRRT) derived from a hereditary renal rat tumor has been used in an assay for initiators and promoters of carcinogenesis based on attachment-independent survival in aggregates. Treatment with single noncytotoxic doses of the carcinogens urethan (1 mM), N-methyl-N-nitrosourea (30 microM), and benzo(a)pyrene (0.2 microM) for 1 hr did not affect survival of HRRT cells in the aggregate assay system. However, when carcinogen treatment was followed by exposure of the cells to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (0.16 microM), a considerable increase in survival was observed. With urethan as an initiator, it was found that tumor promoters (12-O-tetradecanoylphorbol-13-acetate and phorbol-12, 13-didecanoate) induced a considerable response in the assay system, while nonpromoting phorbol esters (4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and 4 alpha-phorbol-12, 13-didecanoate) did not affect the survival. Exposure of HRRT cells to NiSO4 (40 microM) for 3 hr did not influence cell survival in the aggregate form. However, subsequent treatment of the cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate induced a marked increase in the number of viable cells. Moreover, treatment of HRRT cells with a nontransforming dose of urethan (1 mM) for 1 hr followed by continuous exposure to nickel sulfate (40 microM) also increased cell survival in the aggregate form. These results support the view that nickel sulfate may act as both an initiator and a promoter in mammalian cell transformation. The present data also indicate that the aggregation assay system using the HRRT cell line may be a valuable in vitro screening assay for putative initiators and promoters of carcinogenesis.

Leukocyte adherence inhibition assay in human pulmonary neoplasia.

The hemocytometer leukocyte adherence inhibition technique was used to study cell-mediated immuno-activity of patients with lung cancer. KCl extracts (3.5 M) from the lung cancer cell line Calu-1 and the breast cancer cell line MCF-7 were used as antigens. Of 138 patients with lung cancer, 85% showed a positive response against the Calu-1 antigen. The response was independent of the histological type of the tumor and was the same among untreated patients, patients undergoing different types of treatment and patients who died within 3 months after blood collection. Twenty-five percent of the untreated lung cancer patients also reacted against the breast cancer antigen. Among lung cancer patients undergoing different types of treatment, 36% reacted while 50% of the patients who died within 3 months after blood collection reacted against the breast cancer antigen.

Immune responses in lung cancer patients measured by a modified leukocyte adherence inhibition test using serum.

A modified leukocyte adherence inhibition (H-LAI) assay was used to study immunological factors in serum from lung cancer patients. In this test, 0.25% serum was added to the assay system, together with tumor antigen and trypsinized leukocytes from control persons. Extracts from a human lung cancer cell line (Calu-1) and a human breast cancer cell line (MCF-7) were used as antigens. The results obtained were compared with data found with the original hemocytometer (C-LAI) assay. Of 21 lung cancer patients studied, 20 (95%) gave a positive response in both the H-LAI and the C-LAI assay systems against Calu-1 antigen. Only 1 of the patients gave a positive response in the H-LAI system against MCF-7 antigen, while 3 patients (14%) responded in the C-LAI assay. None of the 14 control persons tested gave a positive response. While the C-LAI assay was limited to the use of fresh blood, the H-LAI system was performed on small amounts of serum. The serum could be stored in the frozen state for a long time period. The results indicate that the H-LAI assay possesses at least the same sensitivity and specificity as the original C-LAI test.